Science of the Total Environment 851 (2022) 158259

Contents lists available at ScienceDirect

Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Review

Glyphosate disturbs various epigenetic processes in vitro and

in vivo – A mini review
⁎
Bożena Bukowska a, , Ewelina Woźniak b, Paulina Sicińska a, Katarzyna Mokra a, Jaromir Michałowicz a
a
Department of Biophysics of Environmental Pollution, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska Str. 141/143, 90-236 Lodz, Poland
b
Laboratory of Tissue Immunopharmacology, Department of Internal Diseases and Clinical Pharmacology, Medical University of Lodz, Kniaziewicza 1/5, 91-347 Lodz, Poland

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Glyphosate exhibits epigenotoxicity in

in vitro and in vivo study.
• Glyphosate changes the global methyla-
tion in various cells and organisms.
• Glyphosate changes methylation of differ-
ent promoters of individual genes.
• Glyphosate affects expression of histones
and contributes to expression of various
miRNAs.
• Glyphosate makes transgenerational in-
heritance of F3 pathology in rats.

A R T I C L E I N F O A B S T R A C T

Editor: Henner Hollert Glyphosate in the concentrations corresponding to environmental or occupational exposure has been shown to induce
epigenetic changes potentially involved in carcinogenesis. This substance (1) changes the global methylation in vari-
Keywords: ous cell types and organisms and is responsible for the methylation of different promoters of individual genes, such as
Glyphosate
TP53 and P21 in human PBMCs, (2) decreases H3K27me3 methylation and H3 acetylation and increases H3K9 meth-
Methylation
ylation and H4 acetylation in rats, (3) increases the expression of P16, P21, CCND1 in human PBMCs, and the expres-
Histone
miRNA
sion of EGR1, JUN, FOS, and MYC in HEK293 cells, but decreases TP53 expression in human PBMCs, (4) changes the
Chromatin architecture expression of genes DNMT1, HDAC3, TET1, TET2, TET3 involved in chromatin architecture, e.g. in fish Japanese
DNA methylotransferase medaka, (5) alters the expression of various small, single-stranded, non-coding RNA molecules engaged in post-
transcriptional regulation of gene expression, such as miRNA 182-5p in MCF10A cells, miR-30 and miR-10 in mamma-
lian stem cells, as well as several dozen of murine miRNAs. Epigenetic changes caused by glyphosate can persist over
time and can be passed on to the offsprings in the next generation; in the third generation they can result in some dis-
orders development, such as prostate disease or obesity. Some epigenetic mechanisms have indicated a potential risk
of breast cancer development in human as a result of the exposure to glyphosate. It should be emphasized that the ma-
jority of reported epigenetic changes have not yet been associated with the final metabolic effects, which may depend
on many other factors.

⁎ Corresponding author.
E-mail address: [email protected] (B. Bukowska).

http://dx.doi.org/10.1016/j.scitotenv.2022.158259
Received 10 June 2022; Received in revised form 15 August 2022; Accepted 20 August 2022
Available online xxxx
0048-9697/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/
4.0/).
B. Bukowska et al. Science of the Total Environment 851 (2022) 158259

Contents

1. General information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Influence of glyphosate on various epigenetic processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3. Glyphosate changes global and gene-specific DNA methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.1. Epigenetic mechanisms in glyphosate resistance in plants - increase methylation of EPSP-synthase 1 gene . . . . . . . . . . . . . . . . . . . . 5
4. Glyphosate changes structure of the chromatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4.1. Glyphosate alters expression of methyltransferase and methylcytosine dioxygenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4.2. Glyphosate-induced changes in histone modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4.2.1. Estrogenic activity of glyphosate - changes in the expression of the estrogen receptors, the role of histone modifications . . . . . . . . . . 7
5. Glyphosate alters the level of miRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
5.1. Glyphosate, miRNA, and neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
6. Glyphosate induces epigenetic transgenerational inheritance of pathologies and sperm epimutation . . . . . . . . . . . . . . . . . . . . . . . . . . 8
7. Glyphosate herbicide preparations and epigenetic changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
8. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
CRediT authorship contribution statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Data availability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Declaration of competing interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1. General information et al. (2019) reviewed data from eight published studies evaluating glyph-
osate urinary levels in 423 occupationally and para-occupationally exposed
Glyphosate (N-(phosphonomethyl)glycine) is a total herbicide. Prod- subjects. They showed that average glyphosate urinary levels in occupa-
ucts based on glyphosate are extensively used all over the world to protect tionally exposed subjects varied from 0.26 to 73.5 μg/L (1.53 nmol/L to
agricultural and horticultural crops (Benbrook, 2016). There are over 750 0.43 μmol/L), while environmentally exposed individuals had urinary
different broad-spectrum herbicides containing glyphosate as an active levels from 0.16 to 7.6 μg/L (0.95 nmol/L to 0.045 μmol/L). As a result of
compound (Mesnage et al., 2015). The use of glyphosate-based herbicides intoxication with glyphosate, its level in the human blood was from 0.6
has increased 100-fold from 1974 to 2014 (Vandenberg et al., 2017). to 150 mg/L (3.54 μM - 887.21 μM) (Zouaoui et al., 2013), whereas
Glyphosate is intensively utilized in genetically modified crops, such as soy- during moderate poisoning with glyphosate, its concentrations were in
beans and corn. At present, as much as 56 % of glyphosate is used in genet- the range from 690 mg/L (4.1 mM) to 7480 mg/L (44.2 mM) (Zouaoui
ically engineered herbicide-tolerant crops (Benbrook, 2016). The usage of et al., 2013).
glyphosate-based herbicides is predicted to increase ten-fold in upcoming Epidemiological surveys conducted on humans have shown a possible
years (Benbrook, 2012). Plants that are resistant to glyphosate, metabolize increase in non-Hodgkins lymphomas development in farmers exposed to
it to (aminomethyl)phosphonic acid (AMPA) (Duke and Powles, 2008). glyphosate, while in vivo study proved that glyphosate induced skin
This metabolite exhibits much higher mobility in the soil than glyphosate cancer in mice (George et al., 2010). Research works conducted in vitro
(Annett et al., 2014). Glyphosate is always used along with its adjuvants. have revealed that glyphosate caused oxidative stress, induced DNA
Their role is to enhance glyphosate activity, and therefore contribute to damage, altered estrogen pathway, disturbed brain functions and was
stronger toxicity of glyphosate-based products (Chaufan et al., 2014; implicated in development of various cancer types, including proliferation
Martini et al., 2016). of human breast cancer cells (Marino et al., 2021; Nagy et al., 2019;
Both agriculture workers, as well as humans who do not have direct Peillex and Pelletier, 2020; Portier et al., 2016; Thongprakaisang et al.,
contact with this herbicide, e.g. consumers of products consisting of this 2013).
pesticide residues, are exposed to glyphosate (Chen et al., 2013). In the consequence, the scientists got their attention on the probable ef-
Human is mainly exposed to glyphosate and AMPA present in various fect of glyphosate and its metabolites on the environment and humans. In
crops and food (Vandenberg et al., 2017). Glyphosate residues have also March 2015, the IARC (International Agency for Research on Cancer)
been determned in the samples collected from water, soils, and sediments stated that glyphosate and its products are probable human cancirogens,
(Peruzzo et al., 2008); (Gunarathna et al., 2018; Montiel-León et al., classifying these substances into the Group 2A (IARC Working Group,
2019; Primost et al., 2017; Reynoso et al., 2020), as well as the probes of 2015). However; according to the statement of EFSA (European Food Safety
respirable dust emitted by agricultural soil (Mendez et al., 2017), various Authority) glyphosate is unlikely to be carcinogenic for humans (Tarazona
crops at harvest, and in processed food (Myers et al., 2016; Zoller et al., et al., 2017). The Environmental Protection Agency (EPA) stated in 2017
2018). Food consists of glyphosate in the range of concentrations from that glyphosate was not carcinogenic for humans, while in 2019, EPA re-
trace to 20 mg/kg (barley, soy cereals). As the exposure of humans to glyph- leased a Glyphosate Proposed Interim Registration Review Decision for
osate is still increasing, its levels in edibles are getting bigger. US EPA raised public comments, and in 2020, showed Interim Registration Review
the admissible glyphosate residues level in soy seeds from an initial of Decision, in which it still argues that glyphosate shows no risk to public
2 ppm (by 20 ppm) to as high as 40 ppm (Benbrook, 2016). The average health, if used as indicated (EPA, 2020). In 2017, UE acted to extend the
glyphosate concentration determined in soybean was 3.3 mg/kg (Bøhn glyphosate authorization usage until 2022. Although the consequences of
et al., 2014). During spray application, glyphosate penetrates the human or- the effect of glyphosate on human health ae still uncertain, some countries
ganism mainly by respiratory tract (Clair et al., 2012). decided to take precautionary measures to decrease inappropriate glypho-
Glyphosate has been detected in human blood (mean 73.6 ± 28.2 mg/L sate and glyphosate-based herbicides usage (IARCWorking Group on the
- 435 μM) (Aris and Leblanc, 2011), urine samples: (0.1–3.3 mg/kg bw/ Evaluation of Carcinogenic Risks to Humans, 2017).
day) (Niemann et al., 2015) and (0.5–7.20 ng/mL) (Niemann et al., 2015; Recent studies have shown epigenetic mechanism as a possible media-
Parvez et al., 2018), maternal blood serum (0.2–189.1 ng/mL), umbilical tor of the action of glyphosate and AMPA. In this review, we have described
cord blood serum (0.2–94.9 ng/mL) (Kongtip et al., 2017), and breast the current evidence of glyphosate-induced epigenetic modification in vitro
milk samples (Steinborn et al., 2016) of people who were indirectly ex- and in vivo, and proposed it as potential mechanism through which this
posed to significant amounts of this compound. More recently Gillezeau compound could alter body functions.

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2. Influence of glyphosate on various epigenetic processes 3. Glyphosate changes global and gene-specific DNA methylation

The high rise in glyphosate use over the last years has brought an in- The best-known and the most thoroughly studied epigenetic modifica-
creased concern about its possible toxicity and potential consequences of tion is DNA methylation. It consists of the covalent addition of a
its action to human health. Therefore, evaluation of epigenetic effects of methyl group (-CH3) to the carbon at the position 5 in the cytosine ring,
this herbicide is an important aspect of its toxicity. which leads to the formation of 5-methyl-cytosine (5-mC). Methylation
The definition of epigenetics given by Ben Maamr et al. (2019) is: occurs in cytosines found in CpG dinucleotide moieties. These groupings
‘molecular factors and processes around DNA that regulate genome often appear in clusters to form a series of repeating sequences called CpG
activity, independent of DNA sequence, and are mitotically stable’. islands. CpG islands are most often located in the promoter region of genes
Epigenetic modifications include DNA methylation, post-translational (Jin and Liu, 2018). Gene transcription depends on the availability of the pro-
modifications of histones, and differential expression of non-coding moter and other regulatory regions for transcription factors. DNA methyla-
RNAs. Epigenetic processes may lead to silencing/activating of gene ex- tion reduces the availability of DNA and may inhibit binding of various
pression, genomic imprinting, and pathology development (Carvalho regulatory factors, thus inhibiting gene expression (Portela and Esteller,
et al., 2018; Hemmatzadeh et al., 2020; Nelson et al., 2008). Moreover, 2010). When promoter regions of genes are methylated, transcription factors
epigenetic changes may be maintained over time, and therefore be are not able to bind to them, and the gene expression is decreased. On the
transmitted to offspring in the second, third, and fourth generation contrary, if these sites are demethylated, transcription is activated and gene
(Anway et al., 2005). expression is increased (Rossetti et al., 2020, 2016).
International Working Group of experts of IARC identified 10 key Changes in global methylation level, as well as promoter methylation of
characteristics, one or more of which are usually exhibited by well- genes involved in several essential cellular processes, and the resulting
known human carcinogens. Among these characteristics of carcino- modification of gene expression have very important consequences for eu-
gens are epigenetic changes listed on the 4th position (Smith et al., karyotic cells and organisms. Tumor suppressor genes and proto-oncogenes
2016). Even carcinogens, which do not exhibit genotoxic, apoptotic regulate cell cycle, apoptosis, and cell senescence. Moreover, suppressor
and cytotoxic potential to the cell may still be implicated in carcino- proteins, such as p16, p53, and p21 have important functions in DNA repair
genesis in an epigenetic manner by direct influence gene expression pathways. These pathways are the most frequently compromised in patho-
during transcription, translation, and post-translational events. The logical conditions, such as tumor growth (Kulaberoglu et al., 2016).
epigenetic mechanisms belonging to this special class of carcinogens Inactivating mutations occurring in one or more proteins have been found
yields heritable or non-heritable alterations to the methylation and in most cancer types. Those mutations usually function to reduce progres-
acetylation patterns of DNA and histones. The significance of epige- sion through G1 stage of the cell cycle (e.g.: p16). The proteins, like p53
netics in linking the impacts of environmental factors to alterations function at critical cell-cycle checkpoints, and are responsible for the
in expression of gene is achieving acceptance more and more in recent cycle stopping if a previous step was incorrect or if DNA had been damaged.
years. It is becoming more clear that epigenetic changes play a crucial It has been proven that hypermethylation of promoter regions of p16 and
role in health and disease, including cancer (Chappell et al., 2016; p53 is an epigenetic pattern often reported in human cancer development,
Moggs, 2004; Ors Kumoglu et al., 2022). and this state is connected with a depleted global genomic DNA methyla-
Recent findings have shown that mechanism of action of glyphosate tion (Geraldes et al., 2016).
could comprise epigenetic modifications (Rossetti et al., 2021). These are Changes in total DNA methylation and promoters of TP53 and P16
reversible mechanisms linked to tissue-specific silencing of gene expres- genes under the influence of glyphosate were firstly described by
sion, genomic imprinting, and tumor growth. Particularly, glyphosate has Kwiatkowska et al. (2017). The researchers observed that high concentra-
been reported to produce changes in global DNA methylation, methylation tions of glyphosate (0.25 mM and 0.5 mM) decreased the level of global
of specific genes, histone modification, and differential expression of non- 5mC, and increased methylation of TP53 promoter in human PBMCs. Re-
coding RNAs in human cells and rodents. Importantly, epigenetic changes cently, similar results were reported (additionaly a decrease in methylation
could be herited and could lead to development of a disease long after the of the P21 gene was observed), even at much lower concentrations of glyph-
exposure had ended (Tables 1 and 2). osate of 0.5 μM and 100 μM in human PBMCs (Woźniak et al., 2020a)

Table 1
Changes in global and gene-specific DNA methylation induced by glyphosate.
Type of epigenetic change Observed effects/concentrations of glyphosate References

Global methylation DNA Increase Lung epithelial cells (1500 μM) Ergun and Cayir (2021)
Decrease PBMCs (250 μM and 500 μM) Kwiatkowska et al.
(2017)
PBMCs (0.5 μM and 100 μM) Woźniak et al. (2020a)
MCF10A cells (10−11 M) Duforestel et al. (2019)
Japanese medaka (Oryzias latipes) (0.5 mg/L) Smith et al. (2019)
Changes or not in methylation: Women - 250 CpG sites and 37 regions for which DNA methylation (Lucia, 2021)
was significantly associated with mammographic density
Japanese medaka (Oryzias latipes) – 5727 differentially methylated Smith et al. (2019)
CpG sites between the control and glyphosate at 0.5 mg/L
Rat sperm - an increase of DMRs at 25 mg/kg body weight daily Kubsad et al. (2019)
Pigs – no changes in global methylation of DNA at 200 ppm Larsen et al. (2022)
Rat sperm – DMRs and DHRs in the F3 generation at 25 mg/kg body weight daily Ben Maamar et al. (2021)
Daphnia pulex – an increase or a decrease in methylation in continually Harney et al. (2022)
and switched treatments at 50 μg/L
Single gene promoter methylation Decrease of methylation PBMCs - hypomethylation of TP53 and P21 at 0.5 μM and 100 μM Woźniak et al. (2020a)
and gene expression Changes in gene metylation Sprague-Dawley rats liver - 5727 differentially methylated CpG sites between (Mesnage et al., 2022)
the control and glyphosate at 0.5, 50, 175 mg/kg bw/day for 90 days
Increase in gene expression PBMCs – an increase of P16, P21, CCND1 expression at 0.5 μM and 100 μM Woźniak et al. (2020a)
HEK293 cells – an increase of expression of EGR1, JUN, FOS, and MYC at 0.05–50 μM Jeon et al. (2020)
Decrease in gene expression PBMCs – a decrease of TP53 expression at 0.5 μM and 100 μM Woźniak et al. (2020a)

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Table 2
Other potential epigenetic effects induced by glyphosate.
Type of epigenetic change Observed effects References

Changes in expression of DNMT genes PBMCs – an increase at 0.5 μM and 100 μM Woźniak et al. (2021)
Japanese medaka (Oryzias latipes) – a decrease at 0.5 mg/L Smith et al. (2019)
Pigs - no changes at 20 ppm and 200 ppm Larsen et al. (2022)
Changes in expression of TET genes and increase of its activity Japanese medaka (Oryzias latipes) – an increase in the expression of Smith et al. (2019)
TET1, TET2, and TET3 genes at 0.5 mg/L
Pigs – an increase in expression of TET at 20 ppm and 200 ppm Larsen et al. (2022)
MCF10A cells – an increase of TET 3 activity at 10−11 M Duforestel et al. (2019)
Histone modifications Rats – a decrease of H3K27me3 methylation and H3 acetylation and Lorenz et al. (2019)
an increase of H3K9 methylation and H4 acetylation at 350 mg/kg bw
PBMCs – an increase in the expression of HDAC3 at 0.5 μM and 100 μM Woźniak et al. (2021)
Changes in miRNA level MCF10A cells – increased expression of miRNA 182-5p at 10−11 M Duforestel et al. (2019)
Mammalian stem cell – a decrease of miR-30 and an increase of miR-10 Mesnage et al. (2022)
levels at 0.5, 50, and 175 mg/kg bw/day
Mice - 53 differentially expressed miRNAs at 50 mg/kg/day. Ji et al. (2017)

(Table 1). In other publications, the same authors described a decrease in modifications lasted for 7 months (> 15 generations). The study showed
global DNA methylation in human PBMCs incubated with AMPA at that only some direct alterations in methylation were present in continu-
10 μM and 250 μM (Woźniak et al., 2020a, 2020b). Those authors studied ously exposed populations, while others were found in the organisms
methylation in promoter regions of tumor suppressor genes (P16, P21, both permanently exposed and those transferred to clean water. The re-
TP53) and proto-oncogenes (BCL2, CCND1), and their expression. Impor- searchers also identified modifications in Daphnia pulex that were only pres-
tantly, they found that hypermethylation of TP53 and hypomethylation of ent in populations that had switched to clean water, which indicated a long-
P21 genes after incubation with glyphosate led to changes in their mRNA term legacy of pollutant exposure, which was distinct from persistent
expression. They also noted that glyphosate decreased expression of P16 effects. Differential methylation induced by glyphosate tended to appear
and TP53, as well as increased expression of BCl2, CCND1, and P21, at sites, which were highly methylated in controls organisms. Modifica-
while AMPA increased methylation of TP53 and decreased P21, as well as tions, which were noticed in continuously affected group, as well as in
increased expression of CCND1 (Woźniak et al., 2020a, 2020b). AMPA the group with switched treatments were highly methylated in control indi-
and glyphosate significantly reduced global DNA methylation level in viduals and reveled depleted methylation in the treatments of glyphosate.
human PBMCs, while no changes in the molecular drivers' expression of On the other hand, modifications detected just in the switched treatment
the cell cycle, such as P16, P21, TP53, and BCL2 indicated that AMPA had group tended to possess lower methylation levels in the control and indi-
limited effects on the analyzed epigenetic parameters in human PBMCs cated an increase in methylation in the group with switched treatment.
(Woźniak et al., 2020a, 2020b) (Fig. 1). The most recent study in this area conducted by Larsen et al. (2022)
These disturbances in methylation processes and gene expression in assessed the effect of glyphosate at 20 ppm and 200 ppm (administered
human PBMCs exposed to glyphosate and AMPA, may (as described with feed) on pigs. The authors observed no significant changes in global
above) activate proto-oncogenes, which could lead to genomic alterations, DNA methylation in the kidneys, liver or small intestine of studied animals.
downstream function dysregulation, and a risk of cancer development. However; they revealed that glyphosate in a CpG island of the promoter for
The above results are consistent with reports of Jeon et al. (2020) who IL18 considerably decreased DNA methylation for some individual CpG
showed that glyphosate increased the rate of cell growth in human embry- positions, but these alterations in DNA methylation did not affect on IL18
onic kidney 293 (HEK293) cells. Glyphosate in concentrations ranging from mRNA expression.
0.6 and 18μM was shown to enhance cell proliferation, while other concen- As carcinogenicity and toxicity of glyphosate remains controversial, it
trations of this substance did not alter this process. According to this report, would be interesting to analyze whether epigenetic changes in tumor
glyphosate promoted transcription of EGR1, JUN, FOS, and MYC genes. suppressor genes observed in vivo can be replicated in in vitro models, and
These are critical transcription factors and immediate early genes for cell whether these molecular changes could explain, at least in part, some of
cycle progression, and cell cycle regulators (including cyclin B1, cyclin adverse effects caused by glyphosate.
D1, and p21). Glyphosate increased expression of a long non-coding RNA I has been revealed that methylation of DNA is a marker for exposure to
(NEAT1) associated with cancers and active gene transcription. Thus, it glyphosate and mammographic density that is an essential risk factor for
can be concluded that glyphosate promotes cell proliferation by activating breast cancer development (Lucia, 2021; Lucia et al., 2022). In epidemio-
expression of cell cycle regulatory genes in humans in vitro. logical study, 392 postmenopausal women sent health, diet, and environ-
An opposite effect was observed, i.e. an increase in global methylation mental questionnaires. Moreover, blood and two urinary samples were
under the influence of glyphosate by Ergun and Cayir (2021). They investi- collected from participants of this study. The authors have found that con-
gated molecular mechanisms involved in glyphosate carcinogenicity. The sumption of grain was connected with higher concentrations of glyphosate
level of global m5C DNA methylation and the cytotoxic potential of glyph- in the urine, even in women who consumpted only organic grains (or pre-
osate in A549 lung epithelial cells were determined. Global m5C levels dominantly). Consumption of alcohol, soy and fast-foods have been found
were found to increase significantly in these cell type after exposure to to be linked to higher level of AMPA, whereas consumption of corn and
glyphosate at 1.5 mM for 24 h. A decrease in the DNA methylation due to fruit was due to lower AMPA level (Lucia, 2021). Moreover, 24 CpG sites
glyphosate treatment was also observed by Smith et al. (2019) in the fish methylation was connected with the level of glyphosate in the urine of par-
model of Japanese medaka (Oryzias latipes). Global DNA methylation de- ticipants. The authors of the study combined these sites into a methylation
creased in the developing fry as a result of changes in the expression of index that predicted the concentration of glyphosate in the internal valida-
the methyltransferase and methylcytosine dioxygenases genes of tested tion cohort (Lucia, 2021; Lucia et al., 2022). The calculations have shown
species. that glyphosate and AMPA levels were connected with DNA methylation
Recently, Harney et al. (2022) showed that glyphosate at low concentra- near genes connected with cancer development (SF3B2, MSH4, and
tion of 100 μg/L induced genome-wide differences in methylation of cyto- TRIM31) and endocrine disruption (ESR1), while AMPA level was linked
sine in the freshwater crustacean Daphnia pulex. Uniclonal populations to acceleration of greater epigenetic aging (Lucia, 2021; Lucia et al.,
were permanently exposed to this substance or switched to clean water, 2022). The authors of this study determined 250 CpG sites and 37 regions
and then, methylation was compared to control populations. These for which methylation of DNA was considerably connected with

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Fig. 1. Mechanism of glyphosate-induced changes in the chromatin structure in human PBMCs (Woźniak et al., 2021) (A) and potentially in cell cycle (Woźniak et al., 2020a)
(B). (A) Chromatin plays an essential role in the activation/inhibition of transcription of gene by regulating the binding availability of transcription factors (TFs). Elevated
activity of DNMT1 and HDAC3 leads to condensation of chromatin, preventing the attachment of TFs, which causes inhibition of transcription. It has been shown that
this kind of phenomenon accompanies the process of neoplastic transformation, as it is mainly involved in silencing of expression of suppressor genes. B) A decrease of
gene expression encoding p16 protein that inhibits passage of the cell through checkpoint G1/S phase causes an increase in CCND1 gene expression encoding cyclin D1,
which enables the cell to achieve S phase and contributes to an increase in cell proliferation. In turn, a decrease of the expression of gene encoding p53 protein, which is
an inhibitor of the cell cycle at G2/M phase causes an increase of cyclin B, which leads to activation of cascade of other proteins, and also contributes to cell proliferation.
However; taking into consideration unknown activity of various other proteins involved in the cell cycle regulation, as well as an increase in protein p21 expression
(instead of its decrease) (Woźniak et al., 2020a), which is usually associated with depletion of p53 expression, it is impossible to unequivocally assess (at the current stage
of research) the effect of glyphosate on cell cycle in PBMCs. Nevertheless, the results conducted on other cell types have clearly shown an increase in proliferation of
HEK293 cells (Jeon et al., 2020), Human Skin Keratinocytes HaCaT cells (George and Shukla, 2013), and HepG2 cell line (Kašuba et al., 2017) exposed to glyphosate.

mammographic density (Lucia, 2021). However, they found no statistically women with the highest (in comparison to the lowest) concentrations of
significant association between epigenetic pace of aging and glyphosate or urinary AMPA (Franke et al., 2021).
AMPA levels (Lucia et al., 2022). Exposure to AMPA was connected with ac- Summing this section, numerous studies have shown changes in global
celeration of epigenetic age, which has been previously connected with and gene-specific DNA methylation caused by glyphosate and its metabo-
other environmental exposures (Curtis et al., 2019; Gensous et al., 2020) lite AMPA. The researchers observed both a decrease and an increase, as
and risk of numerous diseases, including breast cancer (Kresovich et al., well as no changes in the above-mentioned parameters (Table 1). It is im-
2019) or B-cell lymphoma (Dugué et al., 2018). Summing up, the exposure portant to note that epigenetic alterations were also noted at very low envi-
to glyphosate and AMPA have been shown to be connected with DNA meth- ronmental concentrations of studied compounds (Jeon et al., 2020; Smith
ylation differences, which could induce cancer development. These results et al., 2019; Woźniak et al., 2020b). Importantly, there are reports in
justified the hypothesis that the exposure to glyphosate and/or AMPA could which glyphosate and AMPA exposure has been associated with differences
increase the risk of cancer. in DNA methylation, which could induce development of breast cancer
The connection of glyphosate and AMPA with differences of DNA meth- (Lucia, 2021; Lucia et al., 2022).
ylation in humans has been rarely investigated. One recent study of pesti-
cide applicators showed a single CpG site (cg06950346) at which 3.1. Epigenetic mechanisms in glyphosate resistance in plants - increase
methylation was connected with history of self-reported glyphosate usage methylation of EPSP-synthase 1 gene
(Hoang et al., 2021).
The above thesis has been confirmed by other studies. Epidemiological Development of weeds' resistance to glyphosate showed different varia-
survey studied the connection between pre-diagnostic excretion of urinary tions in the evolved resistance mechanisms. Plants acquire glyphosate resis-
AMPA and risk of breast cancer in a case-control study of 250 predomi- tance through various mechanisms associated with vacuole herbicide
nantly postmenopausal women: 124 cases and 126 healthy controls. sequestration, rapid response to cell death, nucleotide polymorphisms for
Researchers studied urinary AMPA levels and later breast cancer develop- this herbicide (5-enolpyruvylshikimate-3-phosphate synthase, EPSPS),
ment in Hawaiian women (participating in the Multiethnic Cohort Study) and increased copy number of EPSPS genes. The increased copy number
and found a 4.5-fold increase in risk for developing breast cancer in these of EPSPS is associated with two different genetic mechanisms, i.e., the

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tandem duplication mechanism, and the large extra-chromosomal circular of chromatin architecture, and therefore are able to change patterns of his-
DNA (eccDNA) that is attached to chromosomes and passed on to gametes tone modification, causing alterations in gene expression (Fig. 1).
during meiosis. These divergent mechanisms result in plant dispersal, adap- On the contrary, Duforestel et al. (2019) used non-neoplastic MCF10A
tation to pesticides, and inheritance of resistance (Gaines et al., 2019). cells subjected to repeated glyphosate exposure pattern over 21 days.
Erigeron canadensis (Conyza canadensis, L.) is a weed species that has de- They noted that glyphosate induced a substantial increase in ten-eleven
veloped the resistance to glyphosate. In the study of Tani et al. (2015) the translocation (TET3) enzyme activity and could trigger tumorigenesis of
resistance mechanism on susceptible (S), and resistant (R) populations of breast cells via epigenetic reprogramming that appeared through TET3-
this plant collected from various regions of Greece was assessed. The au- mediated global and local DNA hypomethylation. They showed that
thors evaluated the levels of expression of EPSPS gene, as well as of four glyphosate-mediated DNA hypomethylation is connected with overexpres-
ABC transporter genes, including M10, M11, M7, and P3. The suggested sion of TET3 instead of the DNMT1 pathway. The lower degree of hypome-
mechanism of resistance implicated synchronization of EPSPS and ABC- thylation of DNA reached via the glyphosate-TET3 path in comparison to
transporter genes overexpression. It was suggested that the noticed pheno- that reached via UP peptide-DNMT1 path that is able to induce tumor
typic changes and different expression of EPSPS gene could have been onset may suggest that a great intensity of global hypomethylation of
linked to epigenetic alterations. The findings of this study have proven dif- DNA could act as an oncogenic event, while a moderate intensity of global
ferences in methylation patterns between the two tested populations. It was hypomethylation of DNA might be a predisposing factor to cancer. The in-
reported that nucleotide sequence of the EPSP-synthase 1 gene involved in volvement of TET proteins in breast cancer growth and metastasis has been
the translation start site was more strongly methylated in the R population well-documented (Sun et al., 2013; Yang et al., 2015), and hypomethyla-
in comparison to the S population. Increased methylation of the EPSP- tion of triple-negative breast cancer has been connected with the activity
synthase 1 gene in the R population could have influenced positively its ex- of TET1 DNA demethylase (Good et al., 2018). Notably, Duforestel and
pression, and in the consequence affected positevly on C. canadensis resis- co-workers noticed that siRNA-TET3 abolished glyphosate-induced global
tance to tested herbicide. This study has showed the naturally elevated and local hypomethylation of DUX4 and MTRNR2L2, as well as tumorigen-
resistance of C. canadensis to glyphosate and set the basis for future develop- esis. Duforestel and co-workers analysis with KM plotter database showed a
ment of techniques that may restrict weed resistance to herbicides potentially unfavorable outcome for breast cancers when TET3 is overex-
(Margaritopoulou et al., 2018). pressed. This work revealed that two epigenetic events (global hypomethy-
Summing up, methylation of the EPSP gene in particular is important in lation of DNA and overexpression of a miR) cooperate to induce breast
weed resistance to glyphosate, and knowledge of this process can be useful cancer. These results suggest that TET-specific inhibitor dimethyloxallyl
in overcoming this resistance. glycine (DMOG) might be a reliable therapeutic intervention because
it provides a satisfactory response on both DNA methylation and tumor in-
cidence. This inhibitor acts by limiting TET3-mediated global hypomethy-
4. Glyphosate changes structure of the chromatin
lation of DNA (Duforestel et al., 2019).
Just recently, alterations in the expression of methyltransferase gene
Chromatin structure may be affected by various factors, including
DNMT1 and methylcytosine dioxygenases (TET1, TET2, and TET3) genes
chemicals that can directly target DNA or transcription factors and various
were also determined by Smith et al. (2019) in the fish model of Japanese
binding elements to induce epigenetic changes. The level of chromatin con-
medaka (Oryzias latipes). It was found that glyphosate elevated develop-
densation can significantly influence genes expression and sensitivity of
mental abnormalities in this organism by decreasing of DNMT1 expression
DNA to injury. It is also known that heterochromatin is resistant to DNA
and increasing the expression of TET1, TET2, and TET3. These alterations
damaging agents, while euchromatin has increased sensitivity to damage
led to a reduction in the total DNA methylation. The authors showed that
(Nair et al., 2017).
glyphosate could induce developmental, reproductive, and epigenetic ef-
Glyphosate induces various changes in chromatine structure by
fects in fish.
changing expression of DNMT and TET, as well as altering methylation
As was already mentioned (Larsen et al., 2022) no significant changes in
and acetylation of histones (Table 2).
relative expression of DNMT1, DNMT3A, and DNMT3B genes were noted in
the small intestine, kidney, and liver of pigs exposed to glyphosate at
4.1. Glyphosate alters expression of methyltransferase and methylcytosine 20 ppm and 200 ppm. In contrast, a significant increase in the expression
dioxygenase of TET3 responsible for demethylation, was found in kidneys of these ani-
mals exposed to glyphosate at 200 ppm. Nevertheless, no changes in the
Methylation of DNA is regulated by DNA methyltransferases (DNMT1, global DNA methylation were noticed.
DNMT3A, and DNMT3B), as well as by ten-eleven translocations (TET). In summary, glyphosate causes various alterations in chromatin struc-
Methyltransferases are capable of recognizing unmethylated CpG and ture by changing expression of DNMT, and particularly TET (Larsen et al.,
impart new methylation tags (Lyko, 2018). DNMT1 methyltransferase is ca- 2022; Smith et al., 2019; Woźniak et al., 2021). Glyphosate could induce tu-
pable of recognizing and methylating of hemimethylated CpG dinucleo- morigenesis of breast cells via epigenetic reprogramming that occurred
tides in a process of DNA replication, and is able to replicate methylation through TET3-mediated global and local hypomethylation of DNA. The
pattern into the daughter DNA strands (Jones, 2012). DNMT3A enzymes, fact that active DNA demethylation arranged by TET can appear in resting
which are known as de novo methyltransferases, act to create new patterns (nondividing) cells that represent most of breast cells (in contrast to the ac-
of methylation of hypomethylated DNA, which is crucial for tissue-specific tivity of DNMT that demands cell proliferation) confers to TET-mediated
differentiation during development of the cell (Lyko, 2018). TET mechanism a potentially higher degree of danger for cancer development
enzymes are capable of oxidizing 5-methylcytosine (5-mC) to 5- (Duforestel et al., 2019).
hydroxymethylcytosine (5-hmC), which fnally results in DNA demethyla-
tion in the process of 5-mC elimination from the genome. 4.2. Glyphosate-induced changes in histone modification
Woźniak et al. (2021) reported that glyphosate and its metabolite
AMPA elevated expression of DNA (cytosine-5)-methyltransferase. They Various histones modifications influence chromatin relaxation and
observed that glyphosate at 0.5, 10, and 100 μM elevated expression of packing. The studies concerning diversity of histone modifications and in-
DNMT1, while at 100 μM increased expression of DNMT3. Neverthless, teractions of different modifications have led to the formulation of the his-
glyphosate was not capable of affecting TET 3 gene expression that is tone code hypothesis. It assumes that the pattern of histone modification
known to be able to induce demethylation of DNA. It was also found that determines, which region of the genome is expressed at a given time and di-
glyphosate metabolite - AMPA at 10 μM and 250 μM altered expression of rects aspects, such as regulation of the cell cycle and repair of damaged sites
DNMT1. It is well-known that these enzymes are implicated in regulation in the genome (Miller and Grant, 2013).

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The most common modifications of histone proteins, include acetyla- estradiol. Moreover, this study showed the additive estrogenic effects of
tion (attachment of an acetyl group), methylation (addition of a methyl glyphosate and genistein, which suggested that the usage of glyphosate-
group), phosphorylation (attachment of a phosphate residue) and ubiquiti- contaminated soybean products as dietary supplements may pose a risk of
nation (attachment of the ubiquitin protein). Recently, asparagine breast cancer because of their potential additive estrogenicity. Similarly,
deamidation, arginine, and lysine carbonylation, tyrosine nitrosylation other studies have shown that glyphosate changes the activity of ERα in
and hydroxylation, lysine hydroxylation, cysteine glutathionylation, cancer cells (De Almeida et al., 2018; Sritana et al., 2018).
sulfonylation, glutamine methylation, and H3K4me3 oxidation have also Nevertheless, Mesnage et al. (2017) in the study, which used various
been described (Cavalieri, 2021). molecular biomarkers were not able to replicate the results obtained by
Transcriptional activity and condensation of heterochromatin were Thongprakaisang team. They noticed that glyphosate was capable of acti-
shown to be depleted by trimethylated histone H3K9 (H3K9me3) and vating ERa in vitro, but at its relatively high concentration. The scientists
H3K27me3 (Lee et al., 2020). On the contrary, elevated acetylation of his- noted that Thongprakaisang and co-workers experimental conditions dif-
tones, especially of the H4 tail, was noted to be associated with open, tran- fered to a small degree from those used in their study as their estrogen with-
scriptionally active regions of chromatin (Dhar et al., 2017). The alterations drawal period during cell culture was shorter (1 day vs 5 days). Mesnage
in the structure of chromatin with the DNA methylation, result in activation et al. suggested that difference in the duration of the estrogen withdrawal
or suppression of gene transcription. Woźniak et al. (2021) analyzed the ef- period between the studies should not be a significant factor contributing
fect of glyphosate and AMPA on genes expression participating in the mod- to the observed differences in experimental outcome. The results obtained
ification of histone methylation (EHMT1, EHMT2) and genes involved in by Thongprakaisang and co-workers showing that a concentration of
the modification of histone deacetylation (HDAC3, HDAC5) in PBMCs. An- 10−12 M of glyphosate exhibited a high estrogenic effect raises major con-
alyzed cells were incubated with glyphosate at concentrations ranging from cerns and suggests the possible presence of contaminants. Moreover,
0.5 to 100 μM and with AMPA at concentrations ranging from 0.5 to Mesnage et al. found a surprising fact that Thongprakaisang and colleagues
250 μM. Gene profiling showed that glyphosate (at concentrations of 0.5, were not able to find a no observable effect level for glyphosate in their
10, and 100 μM) and AMPA (250 μM) changed the expression of HDAC3 ERE-luciferase assay (Mesnage et al., 2017).
only. The researchers observed no statistically significant changes in In another study, low dose exposure of glyphosate-based herbicides to
HDAC5, EHMT1, and EHMT2 expression. Moreover, the authors indicated postnatal female rats led to long term changes in mammary gland structure
that glyphosate at lower concentrations than AMPA increased the expres- in adulthood. These alterations included increased percent of mammary
sion of HDAC3. This report clearly showed that glyphosate and AMPA influ- ductal cells with hyperplasia (increased proliferation), as well as morpho-
ence epigenetic processes associated with regulation of chromatin logical alterations characteristic for a pre-cancer state (Zanardi et al., 2020).
architecture. However; unknown activity of many other proteins involved In summary, glyphosate causes long-term epigenetic disruption of the
in the regulation of chromatin structure makes impossible unambiguous uterine ERα gene, which may results in rats implantation failure (Lorenz
evaluation of the effect of tested xenobiotics on the studied process. et al., 2019). Glyphosate is also capable of changing of ERα and ERβ expres-
sion in human hormone-dependent breast cancer, T47D cells in estrogen
4.2.1. Estrogenic activity of glyphosate - changes in the expression of the estrogen withdrawal condition, which may pose a risk of this cancer type develop-
receptors, the role of histone modifications ment (Thongprakaisang et al., 2013). Glyphosate acts through estrogenic
Estrogen receptor alpha (ERα) is crucial for successful embryo implan- pathways, which suggest that exposures during critical periods of breast de-
tation in rats. Lorenz et al. (2019) found that perinatal exposure to a velopment (e.g., gestation, early childhood, adolescence, pregnancy) may
glyphosate-based herbicides induces alterations in this process. Transcrip- lead to deleterious effects on later risk for developing breast cancer.
tion of ERα is known to be under control of five promoters (E1, OT, O,
ON, and OS), which yield different transcripts. These researchers showed 5. Glyphosate alters the level of miRNA
how perinatal exposure to glyphosate changed uterine ERα gene expression
and prompted epigenetic modifications in its regulatory regions during the Short RNAs, named miRNAs act as regulators of gene expression at the
preimplantation period. In the experiment, pregnant rat females (F0) were transcriptional and post-transcriptional levels. Those RNA molecules can
given orally 350 mg of glyphosate/kg bw/day with food, from gestation bind to complementary mRNA (protein synthesis template RNA) and pre-
day 9 until weaning. Glyphosate elevated expression of total ERα mRNA, vent the mRNA from being translated into proteins, and thus inhibit expres-
increasing the abundance of the ERα-O transcript variant. In addition, var- sion. miRNAs are important components of epigenetic machinery and are
ious epigenetic changes were determined in the O promoter. A depletion in themselves subject to epigenetic modification, similar to protein-coding
DNA methylation was noted in one of the three sites evaluated in the O pro- genes. Some research works have unraveled links between expression of ab-
moter. Alterations in methylation and acetylation of histones of the O pro- errant noncoding RNA and human diseases e.g. cancer, cardiovascular and
moter were also found. Histone H4 acetylation and histone H3 lysine 9 neurological disorders (Lekka and Hall, 2018).
trimethylation (H3K9me3) were raised in the O promoter in rats exposed Under normal physiological conditions, miRNAs control biological pro-
to glyphosate, whereas histone H3K27me3 methylation and histone H3 cesses, including the cell cycle, cell proliferation and differentiation, and
acetylation were depleted. The alterations described above may be due to apoptosis. On the other hand, in pathological conditions, deregulation of
observed increase in expression of ERα gene. Perinatal exposure of female a single or small subset of miRNAs has a considerable effect on the expres-
rats to glyphosate caused long-term epigenetic disruption of the uterine sion pattern of several hundred mRNAs. This triggers the cells towards
ERα gene, and may have ultimately been associated with glyphosate- transformation, resulting in development and progression of pathological
induced implantation failure. states (Reddy, 2015).
In another study, Thongprakaisang et al. (2013) assessed the effect of Glyphosate has been revealed to cause a substantial depletion in DNA
glyphosate on expression of estrogen receptors ERα and ERβ on human methylation, and an increase in ten-eleven translocation (TET) 3 activity
breast cancer cells that induce cell proliferation and may cause develop- in MCF10A cells. It was shown that this substance enhanced expression of
ment of cancer. They focused on the effects of pure glyphosate on estrogen miRNA 182-5p (implicated in breast cancer development) and induced
receptors (ERs)-mediated transcriptional activity and their expressions and tumor progression in mice. These results showed that DNA hypomethyla-
found that glyphosate at 10−12 to 10−6 M changed both ERα and ERβ ex- tion appeared via the TET pathway for oncogenic response at presence of
pression and caused proliferative effects only in human hormone- another potential risk factor (Duforestel et al., 2019).
dependent breast cancer, T47D cells in estrogen withdrawal condition. Mesnage et al. (2022) exposed Sprague-Dawley rats to glyphosate at
These results showed that low and environmentally relevant concentrations 0.5, 50, and 175 mg/kg bw/day for 90 days. They noticed that glyphosate
of glyphosate exhibited endocrine-disrrupting activity. The scientists altered genes expression in the liver, which showed TP53 activation by
also observed that glyphosate showed a weaker estrogenic activity than DNA damage and regulation of circadian rhythm. The genes, which the

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activity was the most strongly affected in the liver were similarly to those transgenerational generation animals must cause changes in gene expres-
altered in the kidneys. Low RNA profiling in the liver showed a depletion sion in these animals' somatic cells. In some cases of generational toxicol-
in miR-30, whereas the levels of miR-10 were found to increase. Moreover, ogy, negligible alterations have been seen in the directly exposed
the apurinic/apyrimidinic DNA damage appearred in rat liver along with generations, but increased disease rates are also observed in
increasing exposure to glyphosate. transgenerational descendants (Nilsson et al., 2022). Recently, Kubsad
In summary glyphosate affects mi-RNA levels, it enhances the expres- et al. (2019) showed that glyphosate at 25 mg/kg bw (given daily during
sion of miRNA 182-5p in mice (implicated in breast cancer development) days 8 to 14 of pregnancy caused epigenetic transgenerational inheritance
(Duforestel et al., 2019) and induces a decrease in miR-30 level and an in- of disease and pathology through germline (i.e., sperm) epimutation in fe-
crease in miR-10 levels in rats liver Mesnage et al. (2022). male rats. The authors using a transient exposure of pregnant F0 generation
female rats observed only little effect of glyphosate on the directly exposed
5.1. Glyphosate, miRNA, and neurotoxicity F0 generation and on the F1 offsprings. In contrast, they reported dramatic
increases in pathologies in the F2 generation grand-offsprings, and the F3
Poulsen et al. (2009) proved that glyphosate could cross the blood-brain transgenerational great-grand-offsprings. The following pathologies were
barrier and the placental barrier. In the light of these results, the exposure to reported: obesity, prostate, disorders, ovarian disease, kidney disorders,
glyphosate can lead to neurodevelopmental disorders. Increasing body of as well as birth abnormalities. F1, F2, and F3 generation sperm epigenetic
evidence indicates that miRNAs are dysregulated in numerous pathological analyses showed duplex DNA methylation regions (DMRs). Those re-
states, including neurodevelopmental disorders, such as attention deficit hy- searchers proved that glyphosate can induce the transgenerational inheri-
peractivity disorder (ADHD). Wu et al. (2015) observed that miRNA let-7d tance of disease and germline (e.g. sperm) epimutations.
was elevated in serum of ADHD subjects. Ji et al. (2017) identified miRNA Moreover Ben Maamar et al. (2021) noticed that glyphosate induced
expression patterns in the prefrontal cortex of offspring mice 28 days after epigenetic transgenerational inheritance of pathology and disease in the
birth. Female mice were treated with glyphosate at 50 mg/kg bw/day dur- F3 generation of rats. This research work has demonstrated that differential
ing pregnancy and lactation. The scientists determined 53 miRNAs reveal- histone retention in sperm occurred to play a role in epigenetic
ing different expression than that measured in the control. Among these transgenerational inheritance. These scientists also identified epigenetic
altered miRNAs, there were 11 miRNAs (e.g., miR-34b-5p, miR-19b-3p, biomarkers for glyphosate-induced transgenerational diseases using an
miR-324-5p, miR-320-3p, and miR-322-5p), which are known to be impli- epigenome-wide association study (EWAS). Female rats (generation F0)
cated in brain development and involved in the pathogenesis of were transiently exposed to glyphosate during the developmental period
neurodevelopmental disorders. This study showed that molecular mecha- of gonad sexing. Pregnant rats were transiently exposed (25 mg/kg body
nism of glyphosate-induced neurotoxicity may have been associated with weight glyphosate daily) between days 8–14 of gestation during fetal go-
disregulation of miRNA expression. It was shown that miRNAs could regu- nadal sex determination. Negative effects of the exposure were observed
late target mRNAs, and thus affect the activity of various cells and tissues in the F3 generation (1 year old) without direct exposure. The observed pa-
(Andrés-León et al., 2017). For example, Cell Adhesion Molecule L1 thologies included prostate and kidney disease, obesity, and the presence of
(Chl1), Fibroblast Growth Factor Receptor 1 (Fgfr1), Forkhead Box G1 multiple diseases. Sperm was collected from the glyphosate lineage males
(Foxg1), and Neurontin 1 (Nrn1) are some of the miR-34b-5β target genes affected by a single disease, and used to identify specific differential DNA
may be involved in these processes. These altered genes are known to be in- methylation regions (DMRs), and the differential histone retention sites
volved in a variety of neurological disorders, including autism, schizophre- (DHRs) linked to that pathology. Unique signatures of DMRs and DHRs
nia, and bipolar disorder (Ji et al., 2017; Andrés-León et al., 2017). for each pathology were determined for individual diseases. Interestingly,
Ji et al. (2017) also found several potential miRNA target genes and at a lower statistical threshold, overlapping set of DMRs and DHRs were
their possible neurological pathways. The level of expression of Nuclear Re- identified that were common for all the pathologies. This is one of the
ceptor Subfamily 4 Group A Member 2 (Nr4a2) and Wnt Family Member 7B first observations that sperm histone retention can potentially act as a
(Wnt7b) was downregulated, while Dickkopf WNT Signaling Pathway In- biomarker for specific diseases. DMR and DHR associated genes were iden-
hibitor 1 (Dkk1), DIX Domain Containing 1 (Dixdc1), RUNX Family Tran- tified and correlated with known pathology-specific-associated genes. The
scription Factor 1 (Runx1), sonic hedgehog homolog (Shh), Lymphoid obtained findings allowed to indicate transgenerational epigenetic bio-
Enhancer Binding Factor 1 (Lef-1) and Axin-related protein (Axin2) were markers of disease pathology, which can be found in the sperm that appear
upregulated in the prefrontal cortex of mice offspring following glyphosate to assess disease susceptibility.
exposure during pregnancy and lactation. The majority of the genes men- In other study, perinatal exposure to low doses of a glyphosate-based
tioned above were engaged in regulation of neurogenesis, neuron differen- herbicides injured female reproductive capacity and caused fetal growth
tiation, as well as brain development, showing that these alterations may be delay and structural congenital anomalies in F2 offsprings (Milesi et al.,
essential in the mechanism of glyphosate-induced neurotoxicity (Ji et al., 2018). The exposure of pregnant rats to glyphosate-based herbicides caused
2017). reproductive alterations in the daughter pups of those pregnant dams, and
In summary, molecular mechanism of glyphosate-induced neurotoxicity structural congenital anomalies, retardation in fetal growth and rised pla-
may be connected with disregulation of expression if 11 miRNA, cental weight in the granddaughters (F2 generation) of the dams, even
which have been recognized to be involved in development of brain and though the daughters were only exposed to glyphosate-based herbicides
implicated in the pathogenesis of neurodevelopmental disorders (Ji et al., during gestation. Whether these transgenerational effects would be deter-
2017). mined in modified mammary gland structure and function, as well as risk
for tumor development, remains to be investigated.
6. Glyphosate induces epigenetic transgenerational inheritance of Recently, Harney et al. (2022) determined modifications in glyphosate-
pathologies and sperm epimutation exposed Daphnia pulex and noticed increased methylation in the group
where a switched treatment was applied. The auhors of this study con-
It is well-known that the effect of various environmental toxicants is re- firmed that sublethal doses of glyphosate caused effects in at least three
sponsible for intergenerational inheritance of increased susceptibility to generations following removal of the pollutant.
diseases. It is due, among others, to epigenetic processes, including DNA In summary, glyphosate causes epigenetic transgenerational inheri-
methylation, modification of histones, retention of histones in sperm, tance of diseases and pathologies through germline (i.e. sperm) epimutation
changes in chromatin structure, and expression of non-coding RNAs. The (differential histone retention in sperm) in female rats (Ben Maamar et al.,
exposure to toxicants results in epigenetic alterations in the germ cells 2021; Kubsad et al., 2019). Glyphosate provokes a low or negligible toxic
(sperm or eggs) because germ cells carrying the molecular information to risk at direct exposure, but it may favor generational toxic effects in next
the next generation. In addition, epigenetic alterations that occur in generations in the form of various diseases. Even low doses of glyphosate

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could cause transgenerational epigenetic changes that were consistently that glyphosate and Roundup formulation caused 5727 and 4496 differen-
transferred over many generations (Harney et al., 2022). tially methylated CpG sites, respectively). Small RNA profiling in liver rev-
eled reduced amounts of miR-22 and miR-17 from Roundup MON 52276
7. Glyphosate herbicide preparations and epigenetic changes ingestion. Glyphosate depleted miR-30, while elevated miR-10 levels.
Summarizing several comparative studies of pure glyphosate and its
Numerous studies have shown that herbicide preparations of glypho- preparations, as well as lack of sufficient knowledge on epigenetic effects
sate are more toxic in many ways than pure glyphosate (Chaufan et al., of surfactants used in glyphosate preparations, it is currently impossible
2014; Martini et al., 2016). to answer to the question of whether glyphosate preparation or pure glyph-
Pure glyphosate usually exhibit cytotoxic or other toxic effects at high osate exhibit stronger epigenetic potential.
concentrations, but when used in hrbicide preparations, mainly due to
the presence of surfactants, it shows toxic potential at significanly lower 8. Conclusion
concentrations.
So far, little data has been obtained on epigenetic effects of glyphosate Taking into consideration the widespread and ubiquitous use of glyph-
preparations compared to pure glyphosate, but it is certainly a significant osate in the world, the assessement of the current risk associated with the
problem to be solved. One study showed that glyphosate, but not other exposure to this herbicide is extremely important. The exposure refers not
components present in glyphosate-based herbicides, can induce ERa only to the users of the preparations containing glyphosate, but also to
in vitro, but only at relatively high concentrations. The additives determined humans who do not have direct contact with this pesticide.
in commercial glyphosate-based herbicides formulations were not capable Glyphosate acts through different mechanisms, including dysregulation
of activating of the estrogen pathways in the MCF-7 cells (Mesnage et al., of epigenetic processes (Fig. 2).
2017). On the other hand, other researchers have revealed that develop- A few publications have described epigenetic potential of glyphosate,
mental exposure to glyphosate-based herbicides (from gestational day 9 but the majority of studies have not reported specific biochemical changes
until weaning) causes epigenetic alterations in ERa of F1 rats. They ob- resulting from epigenetic alterations caused by this herbicide. Nevertheless;
served upregulation of the expression of ERa mRNA in uterus of pregnant some specific metabolic effects may not become detectable or may not
rats (Gomez et al., 2019; Lorenz et al., 2019). preced biological effects caused by the exposure to high doses or cumula-
In turn, Smith et al. (2019) demonstrated that in fish Japanese medaka tive long-term exposure to glyphosate. Therefore, further research is
(Oryzias latipes) glyphosate and Roundup exhibit only slight difference in needed in this area.
toxicity as determined by various tested parameters. In another study, Described epigenetic mechanisms shows a potential risk of developing
Roundup at 0.5 mg/L containing glyphosate at reference concentrations breast cancer in humans exposed to glyphosate and its metabolite AMPA.
induced greater changes than pure glyphosate at 0.5 mg/L. Both roundup Duforestel et al. (2019) revealed that glyphosate substantially depleted of
and glyphosate depleted cumulative hatching success. Additionally glypho- DNA methylation in MCF10A cells and accelerated miRNA 182-5p expres-
sate raised developmental anomalities in medaka fry. Moreover, both sion (involved in breast cancer development). They suggested that a great
tested compounds depleted of the expression of the maintenance intensity of global hypomethylation of DNA could act as an oncogenic
DNA methyltransferase gene DNMT1, while they increased the expression event, whereas a moderate intensity of global hypomethylation of DNA
of methylcytosine dioxygenase genes (TET1, TET2 and TET3) in fry at may be taken into consideration as a predisposing factor to cancer. How-
15 dpf, which suggested that epigenetic changes elevayed global DNA de- ever, the final biological effect also depends on a variety of other biochem-
methylation in the developing fry. These results suggest that glyphosate ical factors; therefore it is required to prove this relationship. Epigenetic
and Roundup influence the male reproductive system by modulation of modifications that result from an acute poisoning or chronic environmental
genes needed for spermatogenesis. toxicant exposure is an complex subject where dietary, demographic, and
In recent study, Mesnage et al. (2022) noticed that in rats Roundup behavioral factors play an important role. Recent epidemiological research
formulations induced more biological alterations associated with carcino- works have provided very important information concerning the effect of
genesis than glyphosate. It was also found that both glyphosate and glyphosate on the risk of breast cancer development. Epidemiological stud-
Roundup formulation (MON 52276, European Union) caused epigenetic ies (included 392 postmenopausal women) showed the probability of an in-
modifications in tested model. DNA methylation profiling of liver showed crease in incidence of breast cancer in women exposed to glyphosate

Fig. 2. Glyphosate alters gene regulation, DNA methylation, histone modifications, and the level of miRNA.

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B. Bukowska et al. Science of the Total Environment 851 (2022) 158259

Fig. 3. The effect of glyphosate on various processes connected with a potential increase in the risk of breast cancer development. Glyphosate (1) depletes DNA methylation,
particularly of sites connected with breast cancer (Duforestel et al., 2019; Lucia et al., 2022), (2) increases TET 3 levels and miRNA 182-5p expression (involved in
development of breast cancer) (Duforestel et al., 2019), (3) rises at low concentrations ERα and ERβ expression in human breast cancer cells, as well as induces
proliferative effects only in human hormone-dependent breast cancer cells (Thongprakaisang et al., 2013), (4) increases expression of ERα at relatively high
concentration (Mesnage et al., 2017) and (5) AMPA (glyphosate metabolite) urinary levels correlate with an increased risk of breast cancer development in woman
(Franke et al., 2021).

(glyphosate and AMPA levels were associated with DNA methylation near epigenetic effects of pure glyphosate. It is also important because of the al-
genes connected with cancer development) (Lucia et al., 2022). This obser- most exclusively use of glyphosate preparations and not the pure substance
vation was also confirmed by Multiethnic Cohort Study (included 250 pre- itself.
dominantly postmenopausal women) where 4.5-fold increase in risk of The effect of even low concentrations of glyphosate deregulating epige-
breast cancer development was observed in women with the highest, vs netic mechanisms recognized as a toxic and carcinogenic factor, requires con-
the lowest urinary AMPA concentrations (Franke et al., 2021). The results firmation in vivo models, and especially in epidemiological studies. The long-
of these studies are a very important step in assessing the carcinogenic po- term and multigenerational effects of low doses of glyphosate are also very
tential of glyphosate (Fig. 3). disquieting. The results of the published studies presented in this review con-
The most important data concerning glyphosate epigenotoxicity is stitute a solid base for further analyzes, the more so, hitherho obtained results
consisted in the studies of Ben Maamar et al. (2021) and Kubsad et al. do not allow to answer unequivocally for the question on final health effects
(2019) who noticed that epigenetic effects induced by glyphosate in the F0 associated with glyphosate epiegenotoxicity.
generation of rats are then transferred to the offsprings and their conse-
quences are observed mainly in the third generation, in the form of numerous Funding
disorders, included prostate disease, obesity, kidney disease and others. This
shows the ability of glyphosate to affect on our future generations through This manuscript was funded by Research granted (B2011000000191.01)
epigenetic alterations, and we believe that such changes are just as important to the Department of Biophysics of Environmental Pollution, Faculty of
as the immediate effects of exposure when assessing the risk of chemicals. Biology and Environmental Protection, University of Lodz.
Among the published works, we still have the results that are not un-
equivocally repetitive because of (e.g.) the doses responsible for changes, CRediT authorship contribution statement
for instance, in the estrogen receptor ERα expression (Mesnage et al.,
2017; Thongprakaisang et al., 2013). Bożena Bukowska: Conceptualization, Writing – original draft,
It is also essential to compare the effects of pure glyphosate with its pes- Writing – review & editing. Ewelina Woźniak: Visualization. Paulina
ticide formulations and assess the epigenetic effects of these substances. We Sicińska: Writing – original draft. Katarzyna Mokra: Writing – original
have to find an answer to the question of whether surfactants and other draft. Jaromir Michałowicz: Writing – original draft, Writing – review &
preparation additives accelerate (as in general toxicity) or reduce observed editing.

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B. Bukowska et al. Science of the Total Environment 851 (2022) 158259

Data availability EPA, 2020. https://www.epa.gov.

Ergun, H., Cayir, A., 2021. Exposure to glyphosate and tetrachlorvinphos induces cytotoxicity
and global DNA methylation in human cells. Toxicol. Ind. Health 37, 610–618. https://
No data was used for the research described in the article. doi.org/10.1177/07482337211033149.
Franke, A.A., Li, X., Shvetsov, Y.B., Lai, J.F., 2021. Pilot study on the urinary excretion of the
glyphosate metabolite aminomethylphosphonic acid and breast cancer risk: the multieth-
nic cohort study. Environ. Pollut. 277, 116848. https://doi.org/10.1016/j.envpol.2021.
Declaration of competing interest 116848.
Gaines, T.A., Patterson, E.L., Neve, P., 2019. Molecular mechanisms of adaptive evolution re-
The authors declare that they have no known competing financial inter- vealed by global selection for glyphosate resistance. New Phytol 223, 1770–1775.
https://doi.org/10.1111/nph.15858.
ests or personal relationships that could have appeared to influence the
Gensous, N., Garagnani, P., Santoro, A., Giuliani, C., Ostan, R., Fabbri, C., Milazzo, M.,
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A., Franceschi, C., Bacalini, M.G., 2020. One-year Mediterranean diet promotes epige-
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