This study demonstrates that Roundup reduces JEG3 cell viability at least twice more efficiently than glyphosate. This effect increased with time and was obtained with concentrations of Roundup 10 times lower than that of the agricultural use. The presence of serum buffers the toxic effect of the herbicide. It is generally recognized that serum proteins can bind to chemicals and reduce their availability to cells. Seibert et al. (2002)
have shown that the presence of albumin influences the cytotoxicity of compounds. Moreover, the lack of growth factors in serum-free medium, for instance, could also play a role in this phenomenon. In our experiments, the incubation in serum-free medium was interesting to optimize the visible effects of the compounds in the shortest time. These were also observed anyway after 48 hr in the presence of serum. The physiologic significance of these effects can be questioned, in regard to the concentration used. However, the time of exposure to pollutants may be longer in vivo
, and here in vitro
we observed that long times of exposure allowed low concentrations to present toxic effects. This phenomenon could be caused by metabolism, genomic action, and/or bioaccumulation of some products of Roundup. For instance, Peluso et al. (1998)
demonstrated the formation of covalent links between DNA and some Roundup adjuvants. Their genotoxicity or toxicity was also noticed (Lioi et al. 1998
; Mitchell et al. 1987
; Vigfusson and Vyse 1980
). Even though absorbed Roundup is excreted rapidly from the body, usually in feces (Brewster et al. 1991
; Williams et al. 2000
), a part may be retained or conjugated with other compounds that can stimulate biochemical and physiologic responses. The bioaccumulation of some of its residues may be hypothesized. For example, the harmful effect of glyphosate on semen quality after 6 weeks of post-treatment period in rabbits (Yousef et al. 1995
) may be considered an indication of its retention and conjugation in the body, helped by Roundup adjuvants.
Additionally, in this work Roundup presents a differential time effect at nontoxic levels on aromatase activity of JEG3 cells; this phenomenon was already observed with other xenobiotics such as lindane and bisphenol A (Nativelle-Serpentini et al. 2003
). The 40% rise in aromatase activity after 1 hr of incubation is perhaps caused by an increase of the membrane fluidity and androgenic substrate bioavailability in a first step provoked by adjuvants. By contrast, once well entered into cells, Roundup always reduced aromatase activity. Furthermore, this was associated with the decrease of CYP19
mRNAs. Walsh et al. (2000)
showed that Roundup preferentially diminished the expression of StAR mRNA by decreasing at least the rate of gene transcription.
The direct inhibition of aromatase activity by Roundup was verified in human and equine microsomes, two mammalian aromatase models that we have precisely characterized, in order to understand the active site configuration of this membrane-bound cytochrome P450 (Auvray et al. 1998
; Moslemi and Seralini 1997
; Seralini et al. 2003
). Contrary to results obtained in cells, glyphosate had an inhibitory effect on aromatase activity in human and equine microsomes, but four times lower than the effects of Roundup. Moreover, Roundup inhibited aromatase better in cells than in microsomes (IC50
values, 0.04 and 0.6%, respectively). This could be explained by the difference in incubation duration (18 hr vs. 15 min) inducing metabolism and genomic action. Glyphosate penetration through the cell membrane and subsequent intracellular action appeared in our work to be greatly facilitated by adjuvants, as in plants (Haefs et al. 2002
) or in animal cells, where it can act at the level of cycle regulation (Marc et al. 2002
). Indeed, in this work, minute dilutions of Roundup bringing adjuvants to cells allowed the aromatase inhibitory effect of glyphosate as well as cytotoxic effects.
Moreover, the presence of Roundup in the incubation medium resulted not only in the decrease of the activity of the cytochrome P450 aromatase, but also to a lesser extent in a partial inhibition of its associated reductase. This is confirmed by kinetic and spectral studies that showed that Roundup inhibits the enzyme at the active site level in a competitive manner. Furthermore, our spectral study shows a type II spectrum for purified equine aromatase in the presence of glyphosate or Roundup at the saturating concentration of androstenedione. After androstenedione elimination, Roundup induces a type I spectrum. A type II spectrum with minimal absorbance at 390 nm and maximal absorbance at 420 nm is considered specific for an interaction between a nitrogen atom of the molecule and the heme iron of the cytochrome, whereas a type I spectrum (inverted absorbance) is observed when this type of interaction is absent. Androstenedione, a natural hormone, thus appears to facilitate pesticide access to the active site of the enzyme. However, this occurs more easily with glyphosate directly in contact with the solubilized enzyme than with Roundup, because less concentration of the former was needed to produce the same spectrum.