Perinatal exposure to pesticides alters synaptic plasticity signaling and induces behavioral deficits associated with neurodevelopmental disorders.

Increasing evidence from animal and epidemiological studies indicates that perinatal exposure to pesticides cause developmental neurotoxicity and may increase the risk for psychiatric disorders such as autism and intellectual disability. However, the underlying pathogenic mechanisms remain largely elusive. This work was aimed at testing the hypothesis that developmental exposure to different classes of pesticides hijacks intracellular neuronal signaling contributing to synaptic and behavioral alterations associated with neurodevelopmental disorders (NDD). Low concentrations of organochlorine (dieldrin, endosulfan, and chlordane) and organophosphate (chlorpyrifos and its oxon metabolite) pesticides were chronically dosed ex vivo (organotypic rat hippocampal slices) or in vivo (perinatal exposure in rats), and then biochemical, electrophysiological, behavioral, and proteomic studies were performed. All the pesticides tested caused prolonged activation of MAPK/ERK pathway in a concentration-dependent manner. Additionally, some of them impaired metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD). In the case of the pesticide chlordane, the effect was attributed to chronic modulation of MAPK/ERK signaling. These synaptic alterations were reproduced following developmental in vivo exposure to chlordane and chlorpyrifos-oxon, and were also associated with prototypical behavioral phenotypes of NDD, including impaired motor development, increased anxiety, and social and memory deficits. Lastly, proteomic analysis revealed that these pesticides differentially regulate the expression of proteins in the hippocampus with pivotal roles in brain development and synaptic signaling, some of which are associated with NDD. Based on these results, we propose a novel mechanism of synaptic dysfunction, involving chronic overactivation of MAPK and impaired mGluR-LTD, shared by different pesticides which may have important implications for NDD.

Estrogen receptor-related receptor alpha mediates up-regulation of aromatase expression by prostaglandin E2 in prostate stromal cells.

Estrogen receptor-related receptor alpha (ERRalpha) is an orphan member of the nuclear receptor superfamily of transcription factors. ERRalpha is highly expressed in the prostate, especially in prostate stromal cells. However, little is known about the regulation and function of ERRalpha, which may contribute to the progression of prostatic diseases. We previously found that prostaglandin E2 (PGE2) up-regulated the expression of aromatase in prostate stromal cells. Here we show that PGE2 also up-regulates the expression of ERRalpha, which, as a transcription factor, further mediates the regulatory effects of PGE2 on the expression of aromatase. ERRalpha expression was up-regulated by PGE2 in prostate stromal cell line WPMY-1, which was mediated mainly through the protein kinase A signaling pathway by PGE2 receptor EP2. Suppression of ERRalpha activity by chlordane (an antagonist of ERRalpha) or small interfering RNA knockdown of ERRalpha blocked the increase of expression and promoter activity of aromatase induced by PGE2. Overexpression of ERRalpha significantly increased aromatase expression and promoter activity, which were further augmented by PGE2. Chromatin immunoprecipitation assay demonstrated that ERRalpha directly bound to the aromatase promoter in vivo, and PGE2 enhanced the recruitment of ERRalpha and promoted transcriptional regulatory effects on aromatase expression in WPMY-1. 17Beta-estradiol concentration in WPMY-1 medium was up-regulated by ERRalpha expression, and that was further increased by PGE2. Our results provided evidence that ERRalpha contributed to local estrogen production by up-regulating aromatase expression in response to PGE2 and provided further insights into the potential role of ERRalpha in estrogen-related prostatic diseases.

Enantioselective phytoeffects of chiral pesticides.

Chirality exists extensively in nature. Synthetic chiral plant growth regulators and other pesticides usually behave enantioselectively in phyto-biochemical processes. Chiral plant growth regulators regulate the physiological processes of plants enantioselectively, and chiral pesticides cause enantioselective toxicities or ecotoxicities to plants. On the other hand, these chiral agrochemicals can be absorbed and enantioselectively metabolized by plants. This review summarizes the enantioselective effects of chiral plant growth regulators on plants and the phytotoxic and biotransformation effects of chiral herbicides and several persistent organic pollutants (POPs) on plants. Together, this information on the interactions between chiral agrochemicals and plants might shed light on studies on the chemical and biological behaviors of chiral chemicals, and direct research into the selection of plants, which can potentially decontaminate the environment.

Combined effect of xenoestrogens and growth factors in two estrogen-responsive cell lines.

It is now well recognized that estrogenic signaling mechanisms are far more complex than once thought. Several crosstalks between the estrogen receptor and other signaling pathways may influence the estrogenic stimulation of cell growth. Thus, the estrogenic effects of several environmental contaminants, now suspected to act as endocrine disrupters, may be influenced by a simultaneous stimulation of other signaling pathways. The aim of this study was to investigate whether the growth response of two estrogen-responsive cell lines, MCF-7 and GH3, treated with xenoestrogens might be affected by the addition of growth factors to their culture medium. Cells were treated with two known xenoestrogens, endosulfan and chlordane, alone or in the presence of insulin-like growth factor-1 and epidermal growth factor, respectively, and their growth was measured using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide proliferation assay. Our results show that treatment with endosulfan or chlordane as well as treatment with growth factors increased cell growth, while the administration of xenoestrogens together with growth factors triggered a partly additive response with no antagonist or synergistic effect. These results sustain a role for xenoestrogens in cellular growth.

Effect of tolerance to insecticides on substrate penetration by Formosan subterranean termites (Isoptera: Rhinotermitidae).

Seven selected insecticides were applied to three substrates and bioassayed for barrier efficacy and toxicity against the Formosan subterranean termite, Coptotermes formosanus Shiraki, originating from colonies that differed in their tolerance to the insecticides. A profound substrate effect was seen with all insecticides tested. Sand was the most difficult substrate to penetrate and caused faster and greater mortality of C. formosanus. Increased tolerance in C. formosanus was accompanied by a decrease in penetration of treated substrata with chlordane, methoxychlor, chlorpyrifos, and deltamethrin. The opposite was true in the case of permethrin and bendiocarb. More tolerance C. formosanus displayed decreased mortality in the presence of chlorpyrifos, deltamethrin, bendiocarb, and fipronil. The opposite was true for permethrin.

Modulation of prolactin expression by xenoestrogens.

Xenoestrogens are widely used environmental chemicals that have recently been under scrutiny because of their possible role as endocrine disrupters. Among them are endosulfan and chlordane, two persistent insecticides suspected to act as estrogens in living organisms. To test and better understand the potential estrogenic activity of these chemicals, we used a pituitary cell line (GH(3)) known to respond to estrogens by increasing its secretion of prolactin (PRL), a hormone that is well known for its many physiological functions, especially in fetal growth, development, and reproduction. We measured the levels of PRL secretion and PRL mRNA transcription using immunometric tests, Northern blots, and relative quantitative RT-PCR. We also employed the XTT proliferation assay to compare the growth of GH(3) cells stimulated with 17-beta estradiol and endosulfan or chlordane. Our results show that endosulfan and chlordane are able to induce a substantial increase of PRL expression while these two chemicals do not increase cell growth. Together, our results suggest that endosulfan and chlordane could indeed modulate an estrogen-inducible gene such as PRL, possibly acting via second messenger-mediated cellular mechanisms instead of solely competing with estrogens for the nuclear estrogen receptor sites.

Insecticide susceptibility and detoxication enzyme activities among Coptotermes formosanus Shiraki workers sampled from different locations in New Orleans.

Coptotermes formosanus Shiraki worker termites were sampled from 20 locations in the City Park area of New Orleans, LA. The termites were subsequently assayed to determine their susceptibility to cypermethrin, chlordane and chlorpyrifos, and detoxication enzyme activity. Cypermethrin was most toxic against Formosan subterranean termite workers, chlorpyrifos exhibited intermediate toxicity and chlordane was least toxic. A comparison of insecticide susceptibility between the most and least tolerant colonies revealed 1.9-, 1.7- and 1.8-fold differences in susceptibility for cypermethrin, chlorpyrifos and chlordane, respectively. As with the bioassay data, although significant differences were noted, a great deal of overlap was observed among the colonies for total cytochrome P450 content (difference of 2.2-fold between high and low value) aldrin epoxidation (3.6-fold) and cytosolic esterase (3.9-fold) activity. No significant differences were observed among the colonies for methoxyresorufin O-demethylase or glutathione S-transferase activity. Conversely, microsomal esterase activity varied greatly; a 38-fold difference was observed between the most (Cf1776) and least (Cf1387) active colonies. However, no significant correlation was observed between insecticide susceptibility and microsomal esterase activity. In fact, no significant correlations were observed between any of the enzyme activities measured and insecticide susceptibility. These results are discussed in the context of insecticide selection and future control effectiveness.

Insecticide susceptibility in Coptotermes formosanus and Reticulitermes virginicus (Isoptera: Rhinotermitidae).

Lethal time to mortality responses were established for eight insecticides against workers and soldiers of the Formosan subterranean termite, Coptotermes formosanus Shiraki, and workers of Reticulitermes virginicus (Banks). There were significant differences in the tolerance ratios between workers of C. formosanus colonies to all toxicants tested except fipronil. One colony was 16 times more tolerant than another to deltamethin. C. formosanus soldiers had significant differences in tolerance ratios among colonies exposed to all toxicants except chlorpyrifos. Methoxychlor, permethrin, deltamethrin, and fipronil did not kill soldiers from two, one, one, and three colonies, respectively, within 8 h. Seventy-five percent of R. virginicus colonies were significantly less susceptible than the most susceptible colony to chlordane, methoxychlor, chlorpyrifos, cypermethrin, and fipronil, with 50% of the colonies less susceptible to permethrin and bendiocarb. In 50% of C. formosanus colonies the worker lethal time curves displayed substantial flattening in response to permethrin, and deltamethrin. Lethal time curses for C. formosanus soldiers exposed to chlordane, chlorpyrifos, permethrin, cypermethrin, deltamethrin, and bendiocarb showed substantial flattening. R. virginicus workers demonstrated substantial curve flattening when exposed to chlordane, methoxychlor, chlorpyrifos, deltamethrin, and fipronil. These findings indicate substantial intercolony and intra-colony differences in susceptibility to insecticides.

Role of PKC and MAP kinase in EGF- and TPA-induced connexin43 phosphorylation and inhibition of gap junction intercellular communication in rat liver epithelial cells.

Gap junction intercellular communication (GJIC) is involved in the regulation of many cellular processes. The gap junction channels are made up of connexins and the flow of polar low molecular weight molecules through these channels is inhibited by several groups of substances, such as tumour promoters and growth factors. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), chlordane and the growth factor epidermal growth factor (EGF) are potent inhibitors of GJIC in several cell types, including the rat liver epithelial cell line IAR6.1. The induced inhibition of communication by TPA and EGF in IAR6.1 cells is associated with hyperphosphorylation of connexin43, the connexin responsible for GJIC. Two enzyme inhibitors, PD98059, a specific inhibitor of MEK kinase, and GF109203X, a selective inhibitor of protein kinase C (PKC), were used to study the signalling pathways involved in the effect of EGF and TPA on GJIC, with the following conclusions. The inhibition of cell communication in IAR6.1 cells by EGF is likely to be mediated by direct phosphorylation of connexin43 by MAP kinase. TPA blocks GJIC mainly by the direct action of PKC, but also partly through cross-talk with the MAP kinase pathway. Connexin43 hyperphosphorylation induced by TPA is, as for EGF, mediated through MAP kinase, while PKC seems to block GJIC either through other substrates or induces a type of connexin43 phosphorylation that causes no significant electrophoresis mobility shift.

Quantitative determination of gap junction intercellular communication using flow cytometric measurement of fluorescent dye transfer.

Gap junction intercellular communication (GJIC) is involved in several aspects of normal cell behaviour, and disturbances in this type of communication have been associated with many pathological conditions. Reliable and accurate methods for the determination of GJIC are therefore important in studies of cell biology. (Tomasetto, C., Neveu, M.J., Daley, J., Horan, P.K. and Sager, R. (1993) Journal of Cell Biology, 122, 157-167) reported some years ago the use of flow cytometer to determine transfer between cells of a mobile dye, calcein, as a measure of cell communication through gap junctions. In spite of this being a method with potential for quantitative and reliable determination of GJIC, it has been modestly used, possibly due to technical difficulties. In the present work we have illustrated several ways to use flow cytometric data to express cell communication through gap junctions. The recipient cells were pre-stained with the permanent lipophilic dye PKH26, and the donor cell population were loaded with the gap junction permeable dye, calcein. We show that the method may be used to measure the effect of chemicals on GJIC, and that the information is reliable, objective and reproducible due to the large number of cells studied. The data may give additional information to that obtained with other methods, since the effect observed will be on the establishment of cell communication as compared to what is observed for microinjection or scrape loading, where the effect is on already established communication. This is probably the reason for the more potent effects of DMSO on GJIC measured by the present method than on already existing GJIC measured by microinjection or quantitative scrape loading. We also show that the problem related to the mobile dye calcein not being fixable with aldehydes will not affect the results as long as the cells are kept on ice in the dark and analysed by flow cytometer within the first hours after formalin cell fixation.

Two organochlorine pesticides, toxaphene and chlordane, are antagonists for estrogen-related receptor alpha-1 orphan receptor.

Estrogen-related receptor (ERR) alpha-1 shares a high amino acid sequence homology with estrogen receptor alpha. Although estrogens are not ligands of ERR alpha-1, our recent results suggest that toxaphene and chlordane, two organochlorine pesticides with estrogen-like activity, behave as antagonists for this orphan nuclear receptor. The two compounds increased ERR alpha-1-mediated expression of the reporter enzyme beta-galactosidase in a yeast-based assay. The screen was developed by expressing the hERR alpha-1-yeast Gal 4 activation domain fusion protein in yeast cells carrying the beta-galactosidase reporter plasmid, which contains an ERR alpha-1-binding element. In transfection experiments using mammalian cell lines, such as the SK-BR-3 breast cancer cell line, the compounds were found to have an antagonist activity against ERR alpha-1-mediated expression of the reporter chloramphenicol acetyltransferase. In contrast to the findings with ERR alpha-1, the two compounds were found to slightly induce the estrogen receptor a-mediated expression of chloramphenicol acetyltransferase in SK-BR-3 cells. In a ligand-independent manner, the ERR alpha-1 activity in SK-BR-3 cells was induced 3-fold by cotransfection with the GRIP1 coactivator expression plasmid. Toxaphene was found to be capable of suppressing the GRIP1 coactivator-induced ERR alpha-1 activity in SK-BR-3 cells. In addition, a stable ERR alpha-1 expressing HepG2 hepatoma cell line was generated, and the aromatase activity in the transfected cell line was found to be twice that in the untransfected cell line. The enzyme aromatase converts androgens to estrogens, and aromatase expression in HepG2 cells is regulated in part by an ERR alpha-1-modulating promoter. A 24-h incubation of an ERR alpha-1-transfected HepG2 cell line with 10 microM toxaphene reduced its aromatase activity to the level in the untransfected cell line. Because toxaphene is not an inhibitor of aromatase, it is thought that the decrease of the aromatase activity in ERR alpha-1 transfected HepG2 cells following toxaphene treatment resulted from a suppression of the aromatase expression by toxaphene acting as the antagonist of ERR alpha-1. Toxaphene and chlordane are among the 12 persistent organic pollutants identified by the United Nations Environment Programme as requiring urgent attention. Their antagonistic effects on ERR alpha-1 should not be overlooked.

Suppression of chemokine-induced chemotaxis of monkey neutrophils and monocytes by chlorinated hydrocarbon insecticides.

Chemokines, characterized as pro-inflammatory chemicals made by the immune system, consist of a family of low molecular weight proteins with potent in vitro chemotactic activity causing leukocyte accumulation in vivo. This study determines the effects of organochlorine pesticide exposure on the chemotactic functions of monkey neutrophils and monocytes, using a 48-well chemotaxis chamber. Chemokines IL-8 (interleukin-8) and RANTES were used as the chemoattractants to induce chemotaxis among these monkey leukocytes. Monkey neutrophils or monocytes were first treated with heptachlor, chlordane or toxaphene for 1 hour at 37 degrees C, and the number of cells migrating toward 200 ng/ml IL-8 (for neutrophils) or 100 ng/ml RANTES (for monocytes) were scored. Inhibition of chemotaxis was seen with all samples after treatment with heptachlor, chlordane and toxaphene at concentrations from as low as 10(-14) M to 10(-5) M. Among the three compounds studied, toxaphene was the least effective in preventing monocytes from migrating toward RANTES. The ability of these pesticides to inhibit chemotaxis did not correlate directly with their potential apoptotic effects on the monkey leukocytes. These studies suggest that exposure to organochlorine pesticides may alter leukocyte-related immune functions.

Properties of overlapping EREs: synergistic activation of transcription and cooperative binding of ER.

We have designed a novel estrogen-responsive unit, overERE, which consists of two overlapping ERE separated by 5 bp (center-to-center). In gel retardation assays, this sequence forms a low-mobility complex that migrates like an estrogen receptor tetramer. The receptor-overERE complex was specific and was supershifted by anti-ER H222 antibodies. Dose response studies showed that the formation of the receptor tetramer-overERE complex was cooperative. Truncated receptors were used to assess the contribution of the receptor domains. Deletion of the E domain of the ER prevented the formation of an ER-tetramer complex, which reflects a novel function of this receptor domain. In transfection experiments, 17-beta-estradiol activated transcription from an overERE-containing promoter 4-6 times better than from an ERE-containing promoter. This synergistic effect was observed using either the natural hormone (17-beta-estradiol) or xenoestrogens (phenol red, chlordane). We conclude that two overlapping estrogen-responsive elements can elicit synergistic induction of transcription.

The human acrosome reaction is highly sensitive to inhibition by cyclodiene insecticides.

The mammalian sperm acrosome reaction (AR) is essential to fertilization. It can be initiated in vitro by progesterone, a putative physiological initiator that helps to activate sperm GABA(A) receptor/chloride channels and by glycine, a substitute for the egg zona pellucida, which activates sperm glycine receptor/chloride channels. Even at 1 nM (0.41 ng/ml or 0.41 ppb), chlordane and endosulfan, chlorinated cyclodiene blockers of insect neuronal GABA(A) receptor/chloride channels, strongly inhibited the AR initiated by progesterone or glycine. Inhibition of the latter was also seen at 0.1 nM chlordane and endosulfan, but neither cyclodiene inhibited either AR initiator at 0.01 nM. Inhibitory concentrations of these cyclodienes are well within the range detected in human and wildlife tissue and fluids as a result of environmental contamination.

Synergistic activation of estrogen receptor with combinations of environmental chemicals.

Certain chemicals in the environment are estrogenic. The low potencies of these compounds, when studied singly, suggest that they may have little effect on biological systems. The estrogenic potencies of combinations of such chemicals were screened in a simple yeast estrogen system (YES) containing human estrogen receptor (hER). Combinations of two weak environmental estrogens, such as dieldrin, endosulfan, or toxaphene, were 1000 times as potent in hER-mediated transactivation as any chemical alone. Hydroxylated polychlorinated biphenyls shown previously to synergistically alter sexual development in turtles also synergized in the YES. The synergistic interaction of chemical mixtures with the estrogen receptor may have profound environmental implications. These results may represent a previously uncharacterized level of regulation of estrogen-associated responses.

In vivo prenatal chlordane exposure induces development of endogenous inflammatory macrophages.

Macrophages (m phi s), important cells in host resistance, undergo a series of biochemical changes during their progression from the resident to the fully activated stage. Both resident and inflammatory m phi s are characterized by some unique properties. In the present study, female BALB/c mice were prenatally treated with 8 mg/kg body weight of chlordane, a cyclodiene poly-chlorinated hydrocarbon that appears to reduce immunocompetence by selectively impairing m phi function. Therefore, we examined functions in m phi s from chlordane-treated mice that had been stimulated with thioglycollate. The 5'-nucleotidase activity, present in high levels in resident m phi s but low levels in inflammatory m phi s was elevated in resident m phi s from vehicle-exposed animals. Conversely, inflammatory m phi s from these animals showed significantly diminished levels of this function. Moreover, chlordane-exposed m phi s, regardless of whether they were resident or inflammatory, exhibited decreased 5'-nucleotidase responses. When a second function, transferrin receptor binding, was analyzed, vehicle-treated inflammatory m phi s displayed high levels of activity whereas the resident m phi s showed very little transferrin binding. However, both resident and inflammatory m phi s from the chlordane-exposed group demonstrated transferrin binding activity similar in magnitude to that of the vehicle-treated inflammatory m phi s. Finally, two-dimensional polyacrylamide gel electrophoresis analysis of m phi s from chlordane-exposed mice have characteristics of normal m phi s that have advanced to the inflammatory stage.

Modulation by the insecticides heptachlor and chlordane of the cell-mediated immune proliferative responses of rhesus monkeys.

Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood samples from twelve young, healthy male rhesus monkeys. Triplicate cultures were made in RPMI 1640 with 10% heat-inactivated homologous plasma with or without the test mitogens (phytohemagglutinin, concanavalin A and pokeweed mitogen). Under a defined assay condition, although a different monkey responded to a different degree (3 to 4-fold difference in stimulation index) to a specific mitogen, the proliferative responses of all monkeys were affected significantly by the addition of the chlorinated hydrocarbon heptachlor or chlordane to the test cultures. The insecticides, at 10-40 microM, may also act as mitogens for the unstimulated monkey PBMC or stimulate the release of IL-2 from a mitogen-stimulated culture. At 80 microM, both heptachlor and chlordane completely suppressed the proliferation and IL-2 release of the monkey lymphocytes. These studies suggest immunomodulatory effects of the insecticides heptachlor and chlordane.

Hepatic tumor-promoting chlorinated hydrocarbons stimulate protein kinase C activity.

Various chlorinated hydrocarbons, many of which are known hepatic tumor promoters, have been evaluated for their ability to stimulate protein kinase C (PKC) activity in vitro. Chlordane, kepone, toxaphene, heptachlor, 2,2-bis(4-chlorophenyl)-1,1-dichloroethane, the polychlorinated biphenyl Aroclor 1254, aldrin, 2,2-bis(4-chlorophenyl)-1,1,1-trichloroethane (DDT) and gamma-hexachlorocyclohexane (lindane) were the most potent stimulators of PKC activity. Of these compounds, chlordane was the most potent organochlorine pesticide. Chlordane (100 microM) stimulated mouse brain PKC activity in the 10(5) g supernatant to a maximum velocity equal to that obtained when the enzyme was maximally stimulated with the skin-tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Chlordane concentrations as low as 1 microM significantly stimulated PKC activity. Chlordane-stimulated PKC activity was calcium-dependent, and in the presence of exogenous calcium, chlordane-stimulated PKC activity was at least 5-fold greater than in the absence of added calcium. In contrast, the addition of calcium only minimally affected (less than 30% increase) the TPA-stimulated PKC activity. Concentrations of TPA and chlordane which maximally stimulate PKC did not produce an additive effect on PKC activity. Chlordane- and TPA- stimulated PKC activity was phospholipid-dependent and could be inhibited by quercetin, a known inhibitor of PKC activity. Chlordane in the presence of calcium also stimulated mouse epidermal and hepatic PKC as well as purified rat brain PKC. These results demonstrate that a wide variety of chlorinated hydrocarbons, which are considered hepatic tumor promoters, stimulate protein kinase C activity in vitro.

Expression of cytochrome P450b and P450e genes in small intestinal mucosa of rats following treatment with phenobarbital, polyhalogenated biphenyls, and organochlorine pesticides.

The expression of cytochromes P450b and P450e genes was studied in the small intestinal mucosa of rats using a cDNA which recognizes the mRNAs of both cytochromes as well as oligonucleotide probes which are able to differentiate between the two gene products. Animals were treated with oral and intraperitoneal doses of phenobarbital, gamma-chlordane, trans-non-achlor, and polychlorinated and polybrominated biphenyls. RNA was extracted from small intestinal mucosa and liver. After treatment with each of the compounds, P450b mRNA was markedly induced in small intestinal mucosa and in liver. The greatest degree of induction was found in mucosa of the proximal small intestine where P450b mRNA levels were 4-6-fold higher than levels found in the distal small intestine. This distribution of P450b mRNA was not dependent on the route of administration of inducers. In contrast, although P450e mRNA was induced in the liver after treatment, P450e mRNA in the small intestine did not increase in response to any of the administered inducers. The location of the P450b mRNA within the intestinal mucosa following treatment with inducers was studied by in situ hybridization; the message was induced predominantly in enterocytes located in intestinal villi. These data indicate that the P450b gene is induced in the small intestine following treatment with various xenobiotics and that this induction may be secondary to either transcriptional activation of the gene or to mRNA stabilization in enterocytes located in the villi of the intestinal mucosa. The differential induction of P450b versus P450e genes in the small intestine and liver indicates that the regulation of these closely linked genes is tissue-specific. Furthermore, the marked induction of P450b mRNA in response to the administered xenobiotics indicates that this isoenzyme may have an important biological role in the small intestinal metabolism of environmental toxicants and drugs.