Hepatic CYP1A, 2B, 2C, 2E and 3A regulation by methoxychlor in male and female rats
Introduction
Methoxychlor (MXC; 1,1,1-trichloro-2,2-bis-(4-methoxyphenyl)-ethane), a broad spectrum organochlorine (OC) pesticide, gained popularity because of its short half-life in mammals (Kapoor et al., 1970). However, this compound can be deposited on the ground, bind to soil particles, thus decreasing its mobility (Derr, 1974). MXC is currently used as a substitute for DDT (1,1,1-trichloro-2,2-bis-(chlorophenyl)ethane) which has been banned or strictly regulated since 2000 in industrially developed and other countries due to its toxicity and high persistence in the environment (Matteson and Ramirez, 1999). In Mexico, MXC is presently used for a large number of crops (Matteson and Ramirez, 1999). In spite of the many advantages of MXC use, the widespread application of this pesticide over several decades is expected to result in substantial environmental contamination and, therefore, significant animal and human exposure to both MXC and its metabolites (Li and Kupfer, 1998).
The presence of the methoxy groups in the MXC structure probably accounts for the faster degradation of the compound and its low toxicity in rats (LD50: 6000–7000 mg/kg) which is lower than that of DDT (LD50: 100–250 mg/kg) (Murphy, 1980). However, MXC presents also some undesirable side effects. For example, technical MXC exhibits a higher oestrogenic activity than that observed for the individual isomers (Bitman and Cecil, 1970, Nelson, 1974, Tullner, 1961), as reported both in vivo and in vitro (Bitman and Cecil, 1970, Bulger et al., 1978, Nelson, 1974, Tullner, 1961). In fact, technical MXC induces oestrogen-like changes in the reproductive tract of female rats, thus inhibiting fertility (Gray et al., 1988, Cummings and Gray, 1989, Cummings and Laskey, 1993).
OC pesticides, like DDT and its analogues, are well-known inducers of various hepatic cytochrome P450s (CYP), such as CYP2B1, in rodents (Hart and Fouts, 1965, Kupfer and Peets, 1966, Bunyan et al., 1972). This classifies DDT as a phenobarbital (PB)-type inducer (Lubet et al., 1992). In comparison with DDT, MXC has received little attention, probably since early attempts to provoke a monooxygenase response to MXC exposure in rats met with little or no success. For example, a single low DDT dose (5 mg/kg) increased 1.4-fold the total hepatic CYP content in rats (Sierra-Santoyo et al., 2000), whereas the administration of 200 mg/kg, twice daily for 3 days, resulted in just a 1.1-fold increase (Li et al., 1995). The lack of a significant effect in rats could be due to the rapid biotransformation of MXC in this species (Li et al., 1995). However, multiple dosage treatments with MXC succeeded in inducing rat hepatic CYP (Li et al., 1995).
The reported endocrine disrupting toxicity of DDT in rats and other species (Bulger and Kupfer, 1985, Guillette et al., 1994) could be attributed to its effects on CYPs involved in the metabolism of sex hormones (Sierra-Santoyo et al., 2000). Probably, a similar potential ability could also be attributed to MXC. However, there is no information comparing the influence of sex on the CYP regulation by MXC. Therefore, the aim of this study was to investigate the effect of MXC administration on the expression of different sex-related and other CYPs in rat liver in order to clarify the role of gender, if any, on the response to this pesticide.
Section snippets
Reagents
The following reagents were purchased: 7-ethoxy-, 7-methoxy-, 7-pentoxy, 7-benzyloxy-resorufin, and resorufin from Molecular Probes, Inc., (Eugene, OR); MXC, NADPH and Tris–HCl from Sigma Chemical Co. (St Louis, MO); nitrocellulose paper and other chemicals used for Western blotting from BIO-RAD (Richmond, CA). Goat anti-mouse and anti-rabbit IgG conjugated to horseradish peroxidase were obtained from PIERCE (Rockford, IL) and anti-CYP3A2 from Gentest Corp. (Woburn, MA). Anti-CYP2C11 (Dr. S.
MXC acute toxicity
Any of the MXC doses assayed in this investigation caused significant effects on body weight, food or water consumption. The highest dose used in this experiment represents 4.1–3.6% of the MXC LD50 values mentioned above. However, MXC administration significantly increased the relative liver weight of both male and female rats. Males were affected at all doses tested, while females were effected at the highest doses only (data not shown).
MXC effects on total hepatic CYP content
MXC administration resulted in a dose-dependent increase
Discussion
MXC is known to affect the hepatic CYP-dependent drug metabolizing enzymes (Li et al., 1995). However, the mechanism of this effect, and particularly the role of gender, have not been fully clarified. The results of this study show that MXC induces several CYP-dependent monooxygenases in a sex-specific manner. As a result, the endocrine disruptive effect of MXC may be related with the observed different regulation of CYPs expression in the two sexes. Moreover, the data presented here show that
Acknowledgements
The European Community, EC INCO-DC contract ERB IC18-CT980341, supported the present research. L.F.O.H. was a recipient of a doctoral scholarship from CONACyT, Mexico.
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