Chronic anti-phencyclidine monoclonal antibody therapy decreases phencyclidine-induced in utero fetal mortality in pregnant rats.

Illicit drug use during pregnancy is a serious social and public health problem inflicting an array of deleterious effects on both mother and offspring. We investigated the hypothesis that a murine anti-phencyclidine (PCP) monoclonal antibody (mAb6B5; K(D)=1.3 nM) can safely protect mother and fetus from PCP-induced adverse health effects in pregnant rats. Pregnant Sprague-Dawley rats (n=4-5) were intravenously administered bolus injections of PCP (1mg/kg) on multiple days during pregnancy. They were also chronically treated with anti-PCP mAb6B5 at 45 mg/kg as a PCP antagonist. This dose provided one mAb-PCP binding site for every four PCP molecules. Therapeutic and safety study endpoints included pregnancy outcome (litter size, number of live vs. dead pups), maternal hemodynamic status and locomotor activity. Maternal hemodynamic changes (i.e., blood pressure and heart rate) and locomotor activity were measured in dams from gestation days 6-21 (one day antepartum) using a radiotelemetry-tracking device with a femoral arterial pressure catheter. This mAb6B5 treatment regimen significantly (p=0.008) reduced the number of PCP-induced in utero fetal deaths (odds ratio=3.2; 95%CI 1.3 to 7.9) and significantly (p<0.05) reduced acute PCP-induced maternal locomotor effects in the second trimester. Maternal hemodynamic responses to PCP were not significantly affected by mAb6B5 treatment. In conclusion, these data suggest that anti-PCP mAb treatments administered during pregnancy can safely protect a mother and her fetus(es) from PCP-related morbidity and mortality even when the mAb dose is too low to significantly prevent other PCP-induced maternal pharmacological effects.

Development of a liquid chromatography-tandem mass spectrometric method for the determination of methamphetamine and amphetamine using small volumes of rat serum.

The aim of this paper was to develop LC/MS/MS methodology for the determination of methamphetamine (METH) and amphetamine (AMP) using low microliter volumes (20-150 microl) of rat serum and demonstrate the use of this method for the study of serum pharmacokinetics in the rat. The analytes were extracted from rat serum using solid-phase extraction followed by an isocratic separation on a narrow-bore Hypersil C(18) column. Lower limits of quantitation for METH and AMP were 0.3 ng/ml using positive ion electrospray tandem mass spectrometry. The accuracy of the method was within 20% of the actual values over a wide range of serum concentrations. The within-day and between-day precision was better than 20% (R.S.D.). Ion-suppression matrix effects on electrospray ionization were evaluated for extracted rat serum. The LC/MS/MS method was further validated by comparing serum concentrations of METH and AMP to serum concentrations previously determined using an LC/[ (3)H]-METH assay with radiochemical detection. Finally, the LC/MS/MS method was used to study the pharmacokinetics of METH and AMP after a 1mg/kg intravenous bolus dose of METH to female Sprague-Dawley rats.

Effect of dietary administration of genistein, nonylphenol or ethinyl estradiol on hepatic testosterone metabolism, cytochrome P-450 enzymes, and estrogen receptor alpha expression.

The objective of this study was to examine effects of estrogenic agents of varying potencies (genistein, p-nonylphenol, and ethinyl estradiol) on hepatic testosterone metabolism, cytochrome P-450 (CYP450) enzymes, and ERalpha expression. These endpoints were examined as potential biomarkers of, and contributors to, endocrine disruptive activity. Exposure occurred during critical developmental periods, from gestational day 7 through weaning via the mothers' diet. Thereafter, rats were exposed via their diet to the compounds until puberty (postnatal day 50). Testosterone hydroxylase and 5alpha-reductase activities, CYP2C and CYP3A levels were determined. In general, the compounds were more active in male rats than female rats. The only effect observed in female rats was at the 250 ppm genistein dose, in which an approximately 40% increase in 5alpha-reductase activity was observed. In male rats, genistein treatment had mixed effects on testosterone metabolism. The 1250 ppm dose decreased both CYP2C and CYP3A protein levels. Nonylphenol had the most profound effects on testosterone metabolism and CYP450 expression in male rats, with effects occurring at doses as low as 25 ppm. An increase in 5alpha-reductase activity and a decrease in the formation of 16alpha-OH-, 2alpha-OH-testosterone metabolites, CYP2C and CYP3A protein were observed. EE2 decreased the formation of several testosterone metabolites and CYP2C protein. All compounds had some effect on hepatic ERalpha expression, although a consistent effect was not observed. This study demonstrates that the test compounds can influence hepatic testosterone hydroxylase activity and CYP450 expression, as well as ERalpha expression, although these activities cannot be directly related to estrogenic activity.

Effects of chemical inducers on human microsomal epoxide hydrolase in primary hepatocyte cultures.

Human microsomal epoxide hydrolase (mEH; EC 3.3.2.3) is an important biotransformation enzyme and potential risk determinant for pathologies such as cancer and teratogenesis. Currently, the effects of chemical exposures on human mEH gene expression are largely unknown, but they may constitute a unique modifier of disease susceptibility. To examine this issue, we exposed cultures of primary human hepatocytes isolated from seven donors to prototypic chemical inducers [such as phenobarbital (PB), polyaromatic hydrocarbons, dexamethasone, butylated hydroxyanisole, and ciprofibrate]. Basal levels of mEH RNA and protein were detected readily in untreated cells. Chemical treatment of cultured hepatocytes resulted in variable mEH RNA and protein expression, but, in general, only modest modulatory effects were detected following these exposures. The maximum increase in mEH RNA expression observed was approximately 3.5-fold following Arochlor 1254 exposure. Immunochemical levels of mEH protein were quantified for all treatment groups in three cultures and demonstrated less overall variation and, in general, a lack of concordance with corresponding mEH RNA levels. Cytochrome P450 (CYP) 1A2 and 3A mRNA levels were measured before and following exposure to beta-naphthaflavone and PB, respectively, to permit independent evaluation of hepatocyte inducer responsiveness. Substantial increases in RNA expression levels for both the CYP1A2 and CYP3A genes demonstrated that the hepatocyte cultures were robust and highly responsive to inducer treatment. These results indicate that the mEH gene in human hepatocytes is only modestly responsive to chemical exposures.

Post-transcriptional regulation of human microsomal epoxide hydrolase.

Microsomal epoxide hydrolase (mEH) is a key biotransformation enzyme that is variably expressed in humans. Genetic polymorphisms in the mEH gene have been identified that result in amino acid substitutions in the corresponding enzyme. Results of expression analyses of the mEH allelic variants in vitro suggest that the mutations do not affect the specific activity of the mEH enzyme, but may alter post-transcriptional regulation of mEH. To identify potential post-transcriptional mechanisms that influence mEH expression, the translational efficiency, mRNA half-life, and protein half-life of mEH allelic variants were determined. Constructs encoding each of the four mEH alleles were transcribed in vitro and translated. No differences were detected in the rate of protein synthesis among the variant transcripts, indicating that the previously characterized coding region polymorphisms do not appear to affect translational efficiency. mEH variant RNA half-lives were determined in transfected COS-1 cells, but no differences in decay rates were apparent among the polymorphic constructs. Half-lives of the polymorphic mEH proteins were determined in transiently transfected COS-1 cells treated with the protein synthesis inhibitor cycloheximide. Calculated protein half-lives were: Y113/H139, 15.2 h; H113/H139, 10.7 h, Y113/H139, 16.9 h and H113/R139, 16.0 h. The protein half-lives calculated for the polymorphic variants exhibited the same rank order as mEH protein and activity levels determined previously from expression experiments in vitro and therefore suggest that polymorphic amino acid substitution may result in altered protein stability. However, the differences noted were not statistically significant at the P < 0.05 level, and therefore additional study is required to firmly establish causative relationships.

Metabolism of phencyclidine by human liver microsomes.

These studies examined in vitro metabolism of phencyclidine (PCP) in a series of human liver microsomes (N = 10). Each sample was characterized for cytochrome P450 (CYP) content and for CYP1A, CYP2A, CYP2C, CYP2D, CYP2E, CYP3A, CYP4A, and lauric acid 11-hydroxylation metabolic activities. At least five PCP metabolites (c-PPC, t-PPC, PCHP, an unknown metabolite, and an irreversibly bound metabolite) were formed by the various human liver microsomes. Nevertheless, there was a large degree of inter-individual variation in the metabolite formation. For example, the irreversibly bound metabolite was formed in detectable amounts in only four of the ten samples. c-PPC, t-PPC and the irreversibly bound PCP metabolite formation rates significantly correlated with CYP3A activity. The CYP3A inhibitor troleandomycin was used to inhibit the formation of PCP metabolites. Troleandomycin inhibition was dose dependent with the highest dose producing complete inhibition of the formation of c-PPC, t-PPC, PCHP, and the irreversibly bound metabolite. In addition, PCP inhibited CYP3A-mediated testosterone 6 beta-hydroxylation by 50%. Furthermore, the relative intensity of CYP3A immunoreactive proteins significantly correlated with testosterone 6 beta-hydroxylation and with PCP metabolite formation (except for the unknown metabolite). PCHP formation also correlated with CYP1A activity, while the formation of the unknown PCP metabolite correlated with CYP2A activity. These studies suggest that several CYP isoforms contribute to PCP metabolism and that CYP3A plays a major role in PCP biotransformation in human liver microsomes.

Brain microsomal metabolism of phencyclidine in male and female rats.

These studies examined the microsomal brain metabolism of phencyclidine (PCP) in male and female Sprague-Dawley rats. Several monohydroxylated metabolites of PCP were detected including cis- and trans-1-(1-phenyl-4-hydroxycyclohexyl)piperidine (c-PPC and t-PPC) and 1-(1-phenylcyclohexyl)-4-hydroxypiperidine (PCHP). The in vitro formation of these metabolites required NADPH and was inhibited by carbon monoxide. c-PPC was formed in the male and female brain microsomes at rates of 7.1 +/- 1.3 and 5.7 +/- 1.1 fmol/min per mg, respectively, while t-PPC was formed at rates of 16.2 +/- 3.3 and 16.5 +/- 4.2 fmol/min per mg. PCHP had the highest formation rate at 50.7 +/- 8.9 and 48.2 +/- 8.8 fmol/min per mg, respectively. Although previous studies with rat liver microsomes find higher levels of PCP metabolism in male rats and the formation of an irreversibly bound metabolite in male rats, the present study of brain metabolism found no sex differences in brain metabolism. The formation of PCP metabolites in male rat livers is at least partially mediated by the male-specific isozyme CYP2C11, and possibly CYP2D1. Nevertheless, the formation of the major brain metabolite, PCHP, was not inhibited by an anti-CYP2C11 or an anti-CYP2D6 antibody. However, PCHP formation was inhibited by drug inhibitors of CYP2D1-mediated metabolism, suggesting the involvement of a CYP2D isoform. These data indicate brain metabolism of PCP is significant, but unlike the liver it is not sexually dimorphic.

Human hepatic microsomal epoxide hydrolase: comparative analysis of polymorphic expression.

Interindividual variation in the expression of human microsomal epoxide hydrolase (mEH) may be an important risk factor for chemically induced toxicities, including cancer and teratogenesis. In this study, phenotypic variability and mEH genetic polymorphisms were examined in a bank of 40 transplant-quality human liver samples. Immunochemically determined protein content, enzymatic activities, polymorphic amino acids, as well as mEH RNA levels were evaluated in parallel. Enzymatic activity was assessed using (+/-)-benzo[a]pyrene-4,5-epoxide at 2 substrate concentrations. The relative hydrolyzing activities obtained using saturating substrate levels were highly correlated (r = 0.85) with results derived from limiting substrate concentrations and exhibit approximately an 8-fold range in activity levels across the panel of 40 liver samples. mEH enzyme activity also demonstrated strong correlation (r > or = 0.74) with an 8.4-fold variation determined for mEH protein content within the same samples. However, these protein/activity measurements were poorly correlated (r < or = 0.23) with mEH RNA levels, which exhibited a 49-fold variation. Two common polymorphic amino acid loci in the mEH protein did not exclusively account for variation in enzymatic activity, although this conclusion is confounded by heterozygousity in the samples. These data demonstrate the extent of hepatic mEH functional variability in well-preserved human tissues and suggest that polymorphism of mEH protein expression is regulated in part by posttranscriptional controls, which may include nonstructural regulatory regions of the mEH transcript.

Antipeptide antibodies targeted against specific regions of rat CYP2D1 and human CYP2D6.

Four peptides (pep23-33, pep26-38, pep283-297, and pep409-419) corresponding to unique sequences in rat cytochrome P450 (CYP) 2D1 and/or human CYP2D6 were selected for production of antipeptide antibodies. Rat liver microsomal protein was recognized by anti-serum to all four peptides in ELISAs; however, antisera against pep23-33 and pep26-36 proved not usable for any other applications. Western blots of microsomal protein from a cell line specifically expressing human CYP2D6 revealed that antisera to pep283-297 and pep409-419 recognized a single protein band of the same molecular size as CYP2D6. Antisera to pep283-297 and pep409-419 recognized a rat microsomal protein presumed to be CYP2D1, because it comigrated with human CYP2D6 and had an apparent molecular size of 52 kDa. An unknown protein of approximately 85 kDa was also recognized by pep409-419. Recognition of microsomal protein(s) by antisera to pep283-297 and pep409-419 was blocked by pep283-297 or a bovine serum albumin-pep409-419 conjugate, respectively. Antiserum to pep283-297 was used to analyze sex and strain differences in liver microsomes prepared from Sprague-Dawley, Fischer 344, and Dark Agouti male and female rats. Sprague-Dawley and Fischer 344 rats expressed similar amounts of CYP2D1, but expression in Dark Agouti rats was significantly lower. The antiserum did not detect a sexual dimorphism in any of the strains. A significant correlation between antipeptide283-297 immunoreactivity and Vmax for dextromethorphan O-demethylation existed in female rat strains; however, this relationship did not exist in male rat strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Dose- and time-dependent changes in phencyclidine metabolite covalent binding in rats and the possible role of CYP2D1.

We determined whether chronic dosing with phencyclidine (PCP) could affect the in vitro function of liver microsomal enzymes in male Sprague-Dawley rats. PCP chronic dosing of rats (n = 3 per group) for 3 days with 2.5, 10 and 18 mg/kg/day caused a dose-dependent decrease (23, 36 and 53%, respectively) in the ability of the microsomal enzymes to bind covalently PCP metabolites. The 10- and 18-mg/kg/day dosing groups were significantly different from the 3-day saline-infused control group (P < .05). The results from time-dependent dosing studies indicated PCP covalent binding was significantly reduced (P < .05) in rats (n = 3 per group) infused with 18 mg/kg/day of PCP for 1, 2, 3, 4 and 10 days. Subsequently, it returned to near control values in rats infused for 20 days. In parallel with the time-dependent decreases in covalent binding, the concentrations of at least three phase I PCP mono- and dihydroxylated metabolites were also significantly reduced (P < .05) at the earlier time periods of dosing (3 and 10 days), but the rate of their formation returned to near normal values by 20 days of dosing. Total cytochrome P450 content did not differ from the control groups at any of the doses or time points. As dose- and time-dependent decreases in covalent binding suggested a specific metabolic pathway or isoenzyme was affected, we studied the affect on specific isoenzyme pathways. For these studies a series of cytochrome P450 inhibitors were used.(ABSTRACT TRUNCATED AT 250 WORDS)