Elsevier

Toxicology Letters

Volume 144, Issue 1, 15 September 2003, Pages 93-103
Toxicology Letters

Hepatic CYP1A, 2B, 2C, 2E and 3A regulation by methoxychlor in male and female rats

https://doi.org/10.1016/S0378-4274(03)00230-3Get rights and content

Abstract

The effect on liver cytochrome P450 (CYP) by i.p. injections of methoxychlor (MXC) in corn oil at 0, 100, 150, 200 or 250 mg/kg twice daily for 3 days was investigated in adult male and female Wistar rats. The MXC injection (100 mg/kg b.w.) caused a similar increase of total CYP content in males and females as compared with controls who received the vehicle only. In males, this increase continued up to 250 mg/kg. As to the induction of specific CYP activities, the effect of MXC was found to be sex dependent with three different patterns. Males showed the greatest increases of ethoxy- and methoxyresorufin-O-dealkylase (EROD and MROD, respectively), two CYP1A1/1A2-related activities. On the contrary, females were more responsive than males for pentoxyresorufin-O-dealkylase (PROD) and benzyloxyresorufin-O-dearylase (BROD), two CYP2B-related activities. Finally, p-nitrophenol hydroxylase (PNPH), a CYP2E1-related activity, showed a similar small, although statistically significant, increase for both sexes. As to CYP apoprotein levels, CYP1A1 and CYP2B1/2B2 showed greater increases in females than in males; whereas, interestingly, CYP2E1 induction was higher in males than in females. These results indicate overall that gender modulates CYP expression after MXC injection both qualitatively and quantitatively, and, therefore, this pesticide is not a pure PB inducer. Moreover, the statistically significant increase of CYP3A2 apoprotein expression observed in females and also, to a lower extent, in males, and the decrease of CYP2C11 apoprotein found in males, two sex-related enzymes, may explain the reported endocrine disrupting effect of MXC. The relevance of the different patterns of rat liver CYP induction observed after MXC treatment, in relationship to the speculated endocrine disrupting potential of MXC in humans potentially exposed to this pesticide, needs further investigation.

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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