Review of the earth open source (eos) report " roundup and birth defects: is the public being kept in the dark?"



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A4.3 Cytotoxicity, anti-estrogenic and anti–androgenic activity, and genotoxicity of glyphosate and glyphosate-based herbicides in vitro


Gasnier et al (2009) assessed the activity of glyphosate and four glyphosate-based herbicides (R7.2, R360, R400 and R450; see above evaluation of Benachour and Seralini (2009)) in the HepG2 human hepatoma or MDA-MB453-kb2 cell lines. The following end-points were investigated:

Cytotoxicity: Intracellular SDH activity, extracellular AK activity and intracellular caspase 3/7 activity were measured in HepG2 cells as described by Benachour and Seralini (2009). Cell viability was also assessed by the Alamar Blue assay and the neutral red assay, following 24 hours of exposure to the test compounds over the range 10 – 20 000 ppm.

Anti-oestrogenic activity:

(a) The activity of aromatase, the enzyme responsible for converting androgens to oestrogens, was measured in HepG2 cells after 24 hours of exposure to “non-toxic” concentrations of glyphosate or R7.2, 360, 400 and 450. The assay was based on the release of tritiated water from [1β-H]-androstenedione. Aromatase mRNA levels were also assayed, by semi-quantitative reverse transcriptase-PCR.



(b) Activity at human oestrogen receptors was measured in HepG2 cells transfected with hERα and hERβ and then incubated with 17β-estradiol (at 103-8M) and glyphosate or R7.2 (each at up to 3000 ppm), R360 (up to 2000 ppm), R400 (up to 10 ppm), R450 (up to 30 ppm) or the positive control ICI 182x780 (at 10-8M).

Anti-androgenic activity was measured in MDA-MB453-kb2 human breast cancer cells (which possess a high level of androgen receptor) incubated for 24 hours with glyphosate (up to 1500 ppm) or R7.2, (up to 2000 ppm), R360 (up to 500 ppm), R400 (up to 2 ppm) or R450 (up to 40 ppm) plus DHT (4 X 10-10 M). The positive control was nilutamide (10-6 M).

Genotoxicity: Single- and double-stranded DNA breakage and alkali-labile DNA damage were investigated in HepG2 cells after 24 hours of exposure to R400 at 1, 2.5, 5, 7.5 and 10 ppm, using the single-cell gel electrophoresis (Comet) assay. Benz[a]pyrene (50 µM) was used as positive control. It is unclear whether glyphosate or other Roundup formulations were tested.
Results

Cytotoxicity: SDH and AK activity and the Alamar Blue assay yielded fairly consistent results in the experimental system employed. As shown in the following table, the absolute and relative cytotoxic potencies of glyphosate and Roundup formulations against HepG2 cells were similar to those described by Benachour and Seralini (2009) against other human cell lines in vitro. Again, Roundup formulations were moderately – markedly more toxic than the active constituent, and their relative potency was not proportional to the concentration of glyphosate they contained.

Table 4.4: LOECs or EC50s of glyphosate and Roundup formulations against indices of cytotoxicity in HepG2 cells.

Test compound

Alamar Blue assay

SDH inhibition

AK activity

LOEC (ppm)

EC50*

(ppm)

LOEC (ppm)

EC50*

(ppm)

LOEC

(ppm)

Glyphosate

10 000

27 800

10 000

18 000

>20 000

R 7.2

2000

3600

8000

8600

8000

R360

1000

2200

5000

6500

3000

R400

5

12

50

55

50

R450

50

60

80

170

60

*Reported as LC50

At 60 ppm, R450 formulation induced apoptosis in HepG2 cells, seen as a 156% increase in caspase 3/7 activity following 24 hours exposure (p<0.05 vs control) and a 765% increase after 48 hours (p<0.01). No further data on apoptotic activity were presented.



Anti-oestrogenic activity: Over the range 600 – 3000 ppm, glyphosate had no statistically significant effects on aromatase transcription and activity in HepG2 cells, and was also devoid of anti-oestrogenic activity at hERα and β.

By contrast, intracellular aromatase activity was significantly (p<0.05 or <0.01) inhibited in the presence of Roundup formulations. R7.2 caused ca 75% inhibition at 8000 ppm. R360, R450 and R400 caused no more than ca 50% inhibition of aromatase activity, but maximal inhibition occurred at lower concentrations ( >800, 50 and >10 ppm respectively). The mode of inhibition was not elucidated but is unlikely to have depended on inhibition of DNA transcription, because aromatase mRNA levels were generally increased in Roundup-exposed cells.

All Roundup formulations dose-dependently inhibited oestrogen-dependent transcription in HepG2 cells. R7.2 and R360 were the least potent, with IC50s of ca 1500 – 2500 ppm, whereas R400 and R450 had ca 100 – 500 times greater potency (see following table). Anti-oestrogenic potency was not correlated with the concentration of glyphosate present in the formulations or cell incubation medium.

Anti-androgenic activity: Roundup formulations dose-dependently inhibited androgen-dependent transcription in MDA-MB453-kb2 cells. R7.2 and R360 were the least potent, with respective IC50s of ca 800 and 300 ppm, whereas R400 and R450 had ca 10 – 100 times greater potency (see following table). Anti-androgenic potency was independent of glyphosate concentration.

The study authors claimed that glyphosate “was clearly anti-androgenic at sub-agricultural and non-cytotoxic dilutions”. This is, however, open to question: androgen receptor-mediated transcriptional activity was depressed by ca 30% at the lowest glyphosate concentration tested (100 ppm?), 45% at 500 ppm but only 20% at 1500 ppm (data were presented graphically, so all values are approximate). Although the difference from control was statistically significant (p<0.01) at all three concentrations, the lack of dose-dependency and failure to attain 50% inhibition are remarkable, inconsistent with the behaviour of the Roundup formulations, and seem inconsistent with a receptor-mediated phenomenon. Furthermore, results obtained with the positive control were not presented.



Table 4.5: IC50s of Roundup formulations against human steroid receptors, expressed as ppm formulation (upper line) and µM glyphosate (lower line) in the cell incubation medium

Receptor

R7.2

R360

R400

R450

hERα

2030 ppm

86.5 µM


1450 ppm

3088 µM


6.0 ppm

14.2 µM


20 ppm

53.2 µM


hERβ

2460 ppm

105 µM


1600 ppm

3407 µM


3.0 ppm

7.1 µM


ND

ND


hAR

770 ppm

32.8 µM


310 ppm

660 µM


0.9 ppm

2.1 µM


20 ppm

53.2 µM


hERα = human oestrogen receptor α hERβ = human oestrogen receptor β

hAR = human androgen receptor ND = No data

Genotoxicity: R400 caused a dose-dependent increase in DNA strand breaks16. Compared with the negative control (35% breakage, with 15% class 1, 10% class 2 and 10% class 3 breaks), there was ca 50% total breakage at 5 ppm (comprising 25% class 1, 11% class 2 and 15.5% class 3 breaks), 60% breakage at 7.5 ppm and 75% breakage at 10 ppm (ca 13% class 1, 27% class 2 and 36% class 3 breaks). The NOEC was 2.5 ppm. The positive control caused 95% total breakage, of which ca 70% consisted of class 3 breaks.

However, there results were not necessarily caused by genotoxic activity. In the Alamar Blue assay (the most sensitive index of cytotoxicity), R400 was toxic against HepG2 cells at concentrations of 5 ppm upwards, with an EC50 of 12 ppm. It is therefore possible that the increased DNA strand breakage seen at 5 – 10 ppm arose from cellular injury or death, rather than from direct damage to DNA.



Comment

The study did not demonstrate whether the observed inhibition of aromatase and steroid receptor-mediated transcription was caused by glyphosate or other components in the test products. If surfactants were present, it is highly probable that they contributed to these effects, given that surfactants interfere with in vitro assays for aromatase activity and steroidogenesis (US EPA, 2009; Levine et al, 2007; & DeSesso and Williams, 2012).



Clair et al (2012): The study authors measured the cytotoxicity of glyphosate and a glyphosate-based herbicide, and investigated their effects on testosterone production and oestrogen and androgen receptor mRNA levels in rat testicular cells in vitro. The test compounds were laboratory-grade glyphosate (Sigma-Aldrich, Saint-Quentin Fallavier, France) and Roundup Bioforce (360 g/L glyphosate acid; no other information provided). Stock solutions of glyphosate (7.6 g/L) or 2% Roundup (= 7.6 g glyphosate/L) were prepared in cell culture medium and diluted as required.

Leydig, Sertoli and germ cells were isolated and purified from the testes of 70-day-old Sprague-Dawley rats. Leydig cells were incubated for 1 – 48 hours with Roundup at 0.005 – 1.0% in solution or equivalent concentrations of glyphosate. The other cell types appear to have been exposed to the same range of concentrations for 24 or 48 hours. Cytotoxicity was assessed by measurement of adenylate kinase (AK) activity (an index of cytoplasmic membrane rupture) in cell supernatants using the ToxiLight bioassay. To measure the extent of apoptosis, intracellular caspase 3 / 7 activity was quantified by the Caspase-Glo assay, and nuclear DNA was visualised in situ by DAPI fluorescence staining.

In Leydig cells that had been exposed for 24 hours to glyphosate or Roundup at 0.0001 – 0.10%, 3β-hydroxysteroid dehydrogenase (3β-HSD) activity was measured as an index of testosterone synthesis, and the testosterone concentration in the cell culture medium was quantified by RIA. mRNA expression of aromatase, AR, HERα and HERβ was measured by real-time PCR.

Results

Cytotoxicity (cell lysis): In Leydig cells, glyphosate caused no increase in AK activity over the concentration and time range tested, suggesting a lack of necrosis associated with cytotoxicity. By contrast, cytotoxicity was evident after one hour of exposure to Roundup at >0.10%. The peak effect (ca 3-fold increase in AK activity vs unexposed controls) occurred from 3 – 24 hours at concentrations between 0.50 and 1.0% (p<0.005 or 0.001).

Germ cells were resistant to injury by glyphosate (no increase in AK activity seen) and comparatively insensitive towards Roundup, which caused a maximum of ca 20% increase in AK activity at 24 hours at 0.50% (p<0.001) and at 48 hours at 0.005% (p>0.05).

Glyphosate was cytotoxic towards Sertoli cells, eliciting ca 2-fold increases in AK activity at 24 hours at 0.01 and 0.05% (p>0.05). Roundup also injured Sertoli cells by 24 hours, but the peak effect (a 2-fold increase in AK activity) occurred at 0.10% (p<0.05).

Apoptosis: In the time course experiment with Leydig cells, the only evidence of caspase activation was seen after six hours exposure to Roundup at 0.05%, which elicited a ca 15% increase in activity (p<0.01). Over the 0.1% - 1.0% concentration range, by contrast, Roundup caused concentration-dependent decreases in caspase activity from one hour onwards, with almost complete loss of activity after 12 – 48 hours’ exposure at >0.5% (p<0.001). Roundup caused a similar effect in Sertoli and germ cells after 24 hours of exposure.

In contrast to the formulation, glyphosate did activate caspase in Leydig cells. Relatively weak (10 – 20%) and inconsistent increases in activity were observed from six hours onwards at concentrations of 0.005% and above. In germ cells, 0.005 and 0.01% glyphosate increased caspase activity by ca 20% after 24 hours exposure (p<0.01), while 20 – 40% increases (p<0.01 to 0.001) in activity were evident at 48 hours over the concentration range 0.50 – 1.0%. Glyphosate did not, however, mediate any consistent effect on caspase activity in Sertoli cells.

Morphological evidence of apoptosis (compaction of chromatin and DNA within the nucleus) was observed in Leydig cells exposed for 24 hours to Roundup at 0.05 and 1.0%, or glyphosate at 1.0%. However, there was no comment as to whether nuclear condensation also occurred in Sertoli or germ cells.

Testosterone: Neither Roundup nor glyphosate influenced the 3β-HSD activity in Leydig cells exposed for 24 hours at 0.0001 – 0.10%. Testosterone concentration in the cell incubation medium was depressed by ca 1/3 (p<0.01) by glyphosate and Roundup at 0.0001%, but not at or above 0.005%.

Expression of aromatase, AR, HERα and HERβ in Leydig cells: Aromatase mRNA levels increased by ca 7.5-fold (p<0.005) in response to a 24-hour exposure to glyphosate at 0.001%, but rose by only 2-fold at 0.005 and 0.01% (non-significant). A non-significant, three-fold increase in aromatase mRNA occurred following exposure to Roundup at 0.001%, but at 0.005 and 0.01% there was no effect. Aromatase activity and oestrogen levels were not measured. Neither glyphosate nor Roundup had any effect on androgen or oestrogen receptor mRNA levels under the experimental conditions.

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