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

Luis F. Oropeza-Hernandez, Ricardo Lopez-Romero, Arnulfo Albores *

Seccion de Toxicologıa, Cinvestav-IPN, Avenida IPN 2508, Mexico City 07360, Mexico

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 benzyloxyresor-

ufin-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.

# 2003 Elsevier Ireland Ltd. All rights reserved.

Keywords: Liver CYP; Methoxychlor; Organochlorine pesticides; Sex differences; CYP regulation

1. Introduction

Methoxychlor (MXC; 1,1,1-trichloro-2,2-bis-(4-

methoxyphenyl)-ethane), a broad spectrum orga-

nochlorine (OC) pesticide, gained popularity be-

cause of its short half-life in mammals (Kapoor et

al., 1970). However, this compound can be depos-

ited 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

* Corresponding author. Tel.: �/52-55-5747-3308; fax: �/52-

55-5747-7002/111.

E-mail address: aalbores@mail.cinvestav.mx (A. Albores).

Toxicology Letters 144 (2003) 93�/103

www.elsevier.com/locate/toxlet

0378-4274/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/S0378-4274(03)00230-3

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) (Mur-

phy, 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 cyto-

chrome 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 ofDDT 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). Prob-

ably, a similar potential ability could also be

attributed to MXC. However, there is no informa-

tion comparing the influence of sex on the CYP

regulation by MXC. Therefore, the aim of thisstudy 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.

2. Materials and methods

2.1. Reagents

The following reagents were purchased: 7-

ethoxy-, 7-methoxy-, 7-pentoxy, 7-benzyloxy-re-

sorufin, 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 con-

jugated to horseradish peroxidase were obtained

from PIERCE (Rockford, IL) and anti-CYP3A2

from Gentest Corp. (Woburn, MA). Anti-

CYP2C11 (Dr. S. Bandiera, University of British

Columbia, Vancouver, BC, Canada), anti-CYP1A1, anti-2B1/2B2 and anti-2E1 (Dr. Colin

Jefcoate, University of Wisconsin-Madison, Ma-

dison, WI) were kindly donated to our group.

2.2. Animal treatment

Male and female Wistar rats (2509/50 g) housed

in polycarbonate cages with sawdust bedding were

fed a standard diet (PMI Feed, Inc., St Louis, MO)and water ad libitum, and were maintained at

21 8C and at a 50% relative humidity in a 12 h

dark/light cycle (starting at 07:00 h). Groups of

five rats were injected with MXC (100, 150, 200, or

250 mg of MXC/kg) as a suspension in 0.4 ml of

corn oil, i.p. twice daily for 3 days. A group of rats

L.F. Oropeza-Hernandez et al. / Toxicology Letters 144 (2003) 93�/10394

was similarly injected with corn oil only. Food waswithdrawn immediately after the last MXC or

corn oil injection and rats were killed 24 h later by

deep anaesthesia with pentobarbital. Livers were

perfused with ice-cold saline (NaCl 0.9%, w/v),

excised and processed to obtain microsomes ac-

cording to Mayer et al. (1990). This study was

conducted in compliance with the Mexican Reg-

ulations of Good Laboratory Practice (NOM,2001).

2.3. Other treatments

Positive CYP1A1, CYP2B1/2B2 or CYP2E1

control microsomes were obtained from male

rats treated i.p. with b-naphthoflavone (BNF, 80

mg/kg�/2 days), PB (80 mg/kg�/3 days) orpyridine (Pyr, 100 mg/kg�/3 days), respectively;

and killed under deep anaesthesia with pentobar-

bital.

2.4. CYP content and alkoxyresorufin-O-

dealkylation assays

Total hepatic microsomal CYP content wasmeasured according to Omura and Sato (1964).

Microsomal O -dealkylation of 7-ethoxy- (EROD),

7-methoxy- (MROD), 7-pentoxy- (PROD), and 7-

benzyloxy-resorufin (BROD) were assayed fluor-

imetrically at 37 8C using 530 and 585 nm as the

excitation and emission wavelengths, respectively

(Burke et al., 1985; Lubet et al., 1985; Nerurkar et

al., 1993). The hydroxylation of p -nitrophenol(PNPH) was assayed by the formation of p -

nitrocatechol detected at 510 nm in a Lambda 2S

spectrophotometer (Perkin�/Elmer, Co., Uberlin-

gen, Germany). The PNPH activity was expressed

as nmol of p-nitrocatechol formed per min per mg

of microsomal protein (Reinke and Moyer, 1985).

Proteins were measured according to Lowry et al.

(1951) using bovine serum albumin (BSA) as thestandard.

2.5. Western immunoblots

Microsomal proteins were electrophoretically

separated in SDS-10% polyacrylamide gels (SDS-

PAGE), according to the method of Laemmli

(1970). The proteins were then electrotransferredto a nitrocellulose membrane and the plate

blocked with 100 mM glycine, 1% BSA and 5%

non-fat milk powder overnight at 4 8C. The

membrane was incubated with anti-rat CYP1A1,

2B1/2B2, 2C11, 2E1, or 3A2 antibodies for 1 h,

followed by another 1 h incubation with a horse-

radish peroxidase-conjugated secondary antibody

(goat anti-rabbit IgG). The protein bands werevisualized by chemiluminescence (Amersham, Ar-

lington Heights, IL) followed by a brief exposure

to Kodak XAR Scientific Imaging film. A Foto/

Eclipse videocamera (Fotodyne, Inc.) and Collage

ver. 3.0 software were used to scan the blotted

negatives. Band intensities were compared with the

groups of different treatment.

2.6. Statistical analysis

The analysis of variance (ANOVA) and the

Dunnett’s t-test were used to assess differences

between the experimental groups. A positive

association between any enzyme activities and

protein content was established by linear regres-sion analysis. The level of statistically significant

difference between the mean values was set at P 5/

0.05. All calculations were performed using a

Sigma Stat, ver. 1.0 software (Jandel Corporation,

San Rafael, CA).

3. Results

3.1. MXC acute toxicity

Any of the MXC doses assayed in this investiga-

tion caused significant effects on body weight,

food or water consumption. The highest dose used

in this experiment represents 4.1�/3.6% of theMXC 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).

L.F. Oropeza-Hernandez et al. / Toxicology Letters 144 (2003) 93�/103 95

3.2. MXC effects on total hepatic CYP content

MXC administration resulted in a dose-depen-

dent increase of the total hepatic CYP content.

Similar increases were observed up to the dose of

100 mg/kg for both sexes, whereas CYP content

increased differently at the following doses in

males and females (Table 1). In males, the effect

of MXC started at 100 mg/kg, was dose-dependentand reached a maximum (2.1-fold) at the highest

dose (250 mg/kg), whereas, in females, total

hepatic CYP content increased 1.6-fold at 100

mg/kg and remained constant at the highest doses.

The comparison of the slopes of the MXC effect

on total hepatic CYP content showed a greater

statistically significant response in males (b�/

0.002) than in females (b�/4.13�/10�5) indicatingthat the response depends on gender.

3.3. Effect of MXC on CYP-related activities

MXC administration resulted in a dose-depen-

dent increase of all the CYP-dependent activities

studied in this investigation, regardless of sex. At

the highest MXC dose (250 mg/kg), for example,

EROD, a relatively specific CYP1A1 activity,increased 13.6-fold in males; whereas, a rather

small response was observed in females (4.2-fold)

(Fig. 1A). This observation was in agreement with

the higher calculated slope of the EROD activity

measured after MXC administration in males(b�/35.4), a value 1.8-fold greater than that

observed in females (b�/19.0), the difference

between slopes being statistically significant. On

the other hand, a smaller increase (males, 5.3-fold;

females, 2.3-fold) of MROD activity, a relatively

specific CYP1A2 activity, was observed after

MXC administration. Male rats showed a slope

of b�/19.4 that was 2.6-fold greater than thatobserved in females (b�/7.2) (Fig. 1B).

MXC was an effective inducer of the CYP2B

subfamily dependent enzyme activities PROD and

BROD, since these activities showed even greater

responses than CYP1A-dependent activities. The

dose�/response effect after exposure to MXC

started at 100 mg/kg and reached the maximum

effect at 250 mg/kg both in males and females. Inmales, PROD and BROD activities increased 14.8-

and 3.2-fold, respectively. Notably, the response

was more marked in females than in males, since at

the dose of 250 mg/kg, PROD and BROD

activities were increased 16.4- and 16-fold, respec-

tively, as compared with their respective control

group (Fig. 2A and B). Indeed, slope calculations

for PROD and BROD activities for the doses of100�/250 mg/kg showed different values in male vs.

female animals. The slope for PROD response in

females was steeper (b�/27.0) than that of males

(b�/21.6). A similar pattern was observed for

BROD in females (b�/76.4) and males (b�/46.7).

With both PROD and BROD, sex differences in

the slopes were statistically significant and showed

that females were more responsive than males.As for PNPH, a CYP2E1 dependent activity, a

small but statistically significant increase was

observed. This effect was similar for both sexes

(Fig. 2C). In male rats significant increases were

observed starting from 150 mg/kg, whereas in

females from 200 mg/kg; the maximum effect

(2.0- and 1.8-fold, for males and females, respec-

tively) occurred at the highest dose tested. Theeffect of MXC on the slopes of PNPH activity was

similar in males and females (b�/0.42 and 0.39,

respectively). Thus, sex does not seem to influence

the effect of MXC on CYP2E1 activity.

The MXC induction pattern was different for

males and females in terms of induction potency

and CYP isoform response. In general, CYP1A1-

Table 1

Total hepatic CYP in male and female Wistar rats after MXC

treatment

Dose (mg/kg) Total CYP content (nmol/mg of microsomal

protein)

Male Female

0 0.4509/0.01 0.4109/0.06

100 0.5599/0.02a 0.6529/0.01a

150 0.9269/0.02a 0.5919/0.01a

200 0.9399/0.01a 0.4649/0.02

250 0.9459/0.01a 0.4669/0.02

Values are given as mean9/S.D.; n�/5.a P 5/0.05, male or female vs. the respective control at dose

0. Dunnett?s t -test. See Section 2 for further experimental

details.

L.F. Oropeza-Hernandez et al. / Toxicology Letters 144 (2003) 93�/10396

dependent activities were more responsive in males

than in females. In contrast, CYP2B-dependent

activities were more inducible in females than in

males. Whereas no difference attributed to sex wasobserved in CYP2E1-dependent activity.

3.4. Immunodetection of CYP proteins

The effect of MXC on the immunoreactive

microsomal CYP1A1, 2B1/2B2, 2C11, 2E1, and

3A2 hepatic apoprotein of male and female rats is

shown in Fig. 3. Densitometric analysis of micro-

somal Western blots showed that the CYPs

evaluated followed a sex- and dose-dependent

response. CYP1A1 apoprotein induction was

smaller in males (1.1-fold) than in females (3-

fold) at the highest dose (250 mg/kg), compared

with the control groups (Fig. 3A). In the case of

CYP2B1/2B2, a dose-dependent (100�/250 mg/kg)

induction was observed in all the experimental

groups. These increases were 3.0- and 8.5-fold for

males and females, respectively, as compared with

their respective control group (Fig. 3B). Highly

statistically significant correlations were found

between the two specific apoproteins CYP1A1

and CYP2B1/2B2 and their associated activities

in all groups, i.e. EROD for CYP1A1 and PROD

and BROD for CYP2B1/2B2. The correlation

Fig. 1. Effect of the acute administration of MXC on hepatic microsomal EROD (A) and MROD (B) enzyme activities in Wistar rats.

See Section 2 for experimental details. Values are mean9/S.D. (n�/5). *Statistically significant difference from control group. P �/0.05,

Dunnett’s t -test.

L.F. Oropeza-Hernandez et al. / Toxicology Letters 144 (2003) 93�/103 97

Fig. 2.

L.F. Oropeza-Hernandez et al. / Toxicology Letters 144 (2003) 93�/10398

coefficients between CYP1A1 apoprotein level and

EROD activity in males and females were r�/

0.9485 (P B/0.01) and r�/0.9576 (P B/0.01), re-

spectively.

In males, CYP2B1/2B2 activities and the apo-

protein level showed a slightly better correlation

for PROD (r�/0.9454; P B/0.01) than for BROD

(r�/0.8967; P B/0.01), whereas in females, the

correlations were similar, i.e. r�/0.9933 (P B/

0.01) and r�/0.9817 (P B/0.01), for BROD and

PROD, respectively.

The MXC-dependent increase of the CYP2E1

immunoreactive apoprotein at the highest dose

(250 mg/kg) as compared with the untreated

control was 3.0-fold in males and 1.5-fold in

females (Fig. 3D). These differences in apoprotein

content were not reflected by a corresponding

increase of PNPH activity.

Notably, exposure to MXC decreased the ex-

pression of the male-specific CYP2C11 in a dose-

dependent manner (Fig. 3C), being 0.62-fold that

of the untreated control at the highest MXC dose

(250 mg/kg). Instead, in females, CYP2C11 was

not detected in any group (data not shown).

In addition, MXC also increased the apoprotein

level of the male-specific CYP3A2 in females, the

highest dose used (250 mg/kg) resulting in a 18-fold induction. In contrast, a smaller effect (three-

fold) was observed in males (Fig. 3E). Table 2

summarizes the relative level of induction of the

hepatic CYP apoproteins investigated after MXC

exposure.

4. 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 monooxygenasesin 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 the magnitude of the response in

each gender depends on sex-specific steroidal

Fig. 3. Western blotting analysis of hepatic CYP isoforms obtained from MXC treated rats. Microsomes were subjected to SDS-

PAGE electrophoresis and probed for different CYPs using anti-rat monoclonal antibodies. Ten micrograms of microsomal protein

were loaded on each lane. Lane 1: microsomes from male rats treated with typical inducers, i.e. BNF (CYP1A1) and PB (CYP2B1/2B2

and Pyr (2E1)), or non-treated (CYP2C11, 2E1 and 3A2). Lane 2: microsomes from non-treated animals. Lanes 3�/6: microsomes from

animals treated with 100, 150, 200 and 250 mg of MXC/kg, respectively.

Fig. 2. Effect of the acute administration of MXC on hepatic microsomal PROD (A), BROD (B) and PNPH (C) enzyme activities in

Wistar rats. See Section 2 for experimental details. Values are mean9/S.D. (n�/5). *Statistically significant difference from control

group. P �/0.05, Dunnett’s t -test.

L.F. Oropeza-Hernandez et al. / Toxicology Letters 144 (2003) 93�/103 99

hormones which may be co-involved in the mod-

ulation exerted on the drug metabolism enzymes

by MXC or/and its metabolites. It is possible that

MXC acts as a steroidogenesis regulator or as a

xenoestrogen itself through the activation of the

constitutive androstane receptor system (Blizard et

al., 2001). The modulation of CYPs expression by

MXC may alter the individual capacity to meta-

bolize endogenous and exogenous compounds

such as prostaglandins, drugs, carcinogens and

the steroid hormones themselves (Soucek and Gut,

1992).Interestingly, MXC significantly induced

CYP3A2 expression in a dose-dependent manner

in females (18-fold) as compared with males (3-

fold) and markedly inhibited CYP2C11 in males

(�/38%), as compared with controls. The observa-

tion of the MXC inductive effect on CYP3A2 of

adult female rats is remarkable because CYP3A2

is a male-specific isoform (Waxman et al., 1985).

CYP3A is a gene subfamily composed of multiple

isoforms that have already been characterized by

immunochemistry, catalytic activities, and cDNA

cloning and expression. Hepatic CYP3As, are

responsible for the enzyme oxidation of numerous

clinically used drugs, and this subfamily is induced

by a variety of compounds, including naturally

occurring and synthetic glucocorticoids (e.g., dex-

amethasone), pregnane compounds (e.g., pregne-

nolone 16-carbonitrile), and macrolide antibiotics

(e.g., rifampicin) (Quattrochi and Guzelian, 2001),

among others. Although CYPs 3A23 and 3A2

share a high degree of structural homology, they

are regulated independently (Telhada et al., 1992).

For instance, dexamethasone is a more potent

inducer of CYP3A23 than PB. In contrast,

CYP3A2 is induced only by PB (Ghosal et al.,

1996; Gonzalez et al., 1986). The increase of

CYP3A2 observed here confirms the findings of

Li et al. (1995) who reported increases in the

hydroxylation of testosterone at the 6b, 2b and

15b positions, suggesting that MXC enhances

CYP3A apoprotein level and its related activities.

MXC diminished the CYP2C11 apoprotein level

in male rats, an observation in agreement with the

mechanism proposed by Akingbemi et al. (2000) of

the participation of 2,2-bis-(p-hydroxyphenyl)-

1,1,1-trichloroethen, an MXC metabolite, in the

reduction of testosterone biosynthesis in rat Ley-

dig cells by suppressing the mRNA steady-state

and the enzyme cleavage of the cholesterol side-

chain, thus suggesting that MXC may act as an

anti-androgen. Furthermore, a marked decrease of

2a- and 16a-testosterone hydroxylation was ob-

served when liver microsomes from PB male

treated rats were co-incubated with MXC and

testosterone, again indicating a strong inhibition

of MXC on CYP2C11-related enzyme activity (Li

et al., 1993).

Notably, MXC treatment-induced CYP1A1/

1A2, as measured by their associated enzyme

activities EROD and MROD and by their apo-

protein content, being males more responsive than

females. The differences between the relative

increase of the apoprotein content and the asso-

ciated enzyme activities observed in this study for

the CYP1A subfamily in male and female rats are

consistent with previous reports for the analogues

Table 2

Relative induction of hepatic CYP isoforms after an MXC treatment in male and female Wistar rats

Dose (mg/kg) 1A1 2B1/2B2 2C11 2E1 3A2

M F M F M M F M F

0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

100 0.4 0.6 0.9 1.3 0.80 1.2 0.5 1.3 2.2

150 0.5 0.9 1.2 2.5 0.72 1.5 0.8 1.8 4.5

200 0.6 1.5 1.8 4.5 0.66 2.0 1.0 2.3 7.0

250 1.1 3.0 3.0 8.5 0.62 3.0 1.5 3.0 18.0

Note: Relative induction�/exposed group/control group. Immunoreactive protein was determined in liver microsome samples

pooled for each group. See Section 2 for details.

L.F. Oropeza-Hernandez et al. / Toxicology Letters 144 (2003) 93�/103100

of MXC, DDT or PCBs (Sierra-Santoyo et al.,2000; Flodstrom et al., 1990). The MXC-depen-

dent induction of the CYP1A subfamily apopro-

teins and related activities contrasts with the

observations by Li and Kupfer (1998) who found

no increase of the mRNA levels of CYP1A1.

However, no attempt was made by these authors

to measure the apoprotein content and the related

enzyme activity. Taking these observations to-gether it is possible to suggest that MXC regula-

tion of CYP1A is posttransductional rather than

posttranscriptional. Other chemicals, like the

MXC analogue DDT, are also able to increase

EROD activity without affecting the correspon-

dent apoprotein content (Sierra-Santoyo et al.,

2000), thus supporting the fact that CYP1A may

be regulated at different levels.In contrast, the levels of CYP2B1/2B2 apopro-

tein and its associated enzyme activities (PROD

and BROD) showed that female rats to be more

responsive to MXC than males. In addition, both

females and males displayed a greater response of

PROD than of BROD activities. These results are

in agreement with those reported by Li et al.

(1995) showing that CYP2B1/2B2 apoproteinswere induced in mature female rats. In addition,

these authors suggested that MXC increased the

16a- and 6b-hydroxylation of testosterone and

androstenedione formation, indicating an MXC-

mediated induction of CYP2B enzyme activities.

Although we could not find any studies on MXC

reproductive toxicity in humans, several studies in

animals, both males and females, suggest thatMXC is able to affect the reproductive system

(Bulger and Kupfer, 1985). These effects are

thought to be due to the oestrogenic activity of

both O -demethylated metabolites of MXC and

some of the O -demethylated contaminants of

technical grade MXC (Bulger and Kupfer, 1985),

CYP2B1 being the isoform responsible for the O -

demethylation and ring-hydroxylation of MXC(Dehal and Kupfer, 1994).

The inductive pattern of MXC on CYP reported

here mimics that observed for DDT (Sierra-

Santoyo et al., 2000). Although MXC showed to

be an effective inducer in both sexes, a larger dose

and a repetitive exposure scheme were needed with

MXC to reach a similar level of induction to that

caused by a small single dose of DDT (Sierra-Santoyo et al., 2000).

In summary, our results showed that MXC is a

relatively weak, but still effective, inducer of CYP

and related activities. The magnitude of the

response to this pesticide depends on gender and

involves sex-specific isoforms. The preferential

induction observed for CYPs involved in steroid

metabolism (2B1, 2B2 and 3A2) implies that MXCmay be able to change the pattern of steroid

turnover. In addition, the MXC-mediated induc-

tion of the CYP1A subfamily suggests that this

pesticide is not a pure PB inducer. Taken together

these results indicate that the induction by MXC

of several CYP isoforms in rat liver is capable of

altering the hormonal homeostasis in this species.

However, the mechanisms responsible for thegeneral and gender-specific modulation by MXC

of the expression of different CYP isoforms in rats

and the relevance of these events for humans

remain to be established.

Acknowledgements

The European Community, EC INCO-DC con-

tract 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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