Patricia Linko

Expression of Cyp1a1 and Cyp1a2 genes in human liver

Immunoblot analysis of human livers using a monospecific antibody to rat CYP1A2 section demonstrated that the expression of CYP1A2 protein is highly variable in human liver. Quantitative PCR analysis was then employed to examine the interindividual variability of both CYP1A1 and CYP1A2 mRNAs in human liver. Hepatic content of CYP1A2 mRNA correlated significantly with levels of CYP1A2 protein as analysed by immunoblot analysis (r = 0.58; p < 0.01). CYP1A2 mRNA content varied > 40-fold among individuals while CYP1A1 content varied > 20-fold. CYP1A2 mRNA was higher than CYP1A1 mRNA (approximately two to 30-fold) in livers of different individuals. The individual with the highest CYP1A1 and CYP1A2 mRNA amounts was a current smoker, but mRNA expression in two other smokers was within the range observed among nonsmokers. The expression of the two CYP1A mRNAs correlated highly (r = 0.72; p < 0.0005) when smokers were included, but the correlation was less significant (r = 0.62; p < 0.05) in nonsmokers. We amplified a full-length CYP1A2 cDNA clone by PCR from a liver which expressed extremely low amounts of CYP1A2 protein. Sequence analysis indicated that exon 4 was missing in this clone, but no other sequence changes were found. PCR analysis demonstrated that both the normally spliced mRNA and abnormally spliced mRNA could be detected in all human livers examined, but the normally spliced mRNA was more abundant than the splice variant. Therefore, sequence changes in the coding region of CYP1A2 did not account for the poor expression of CYP1A2 in this individual.

Interaction of hexachlorobenzene with the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in vitro and in vivo,: Evidence that hexachlorobenzene is a weak Ah receptor agonist☆☆☆

Hexachlorobenzene (HCB) produces hepatic porphyria and induces the hepatic cytochrome P450 isozymes P450c (P450IA1) and P450d (P450IA2) in rodents. These and other effects of HCB resemble those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which acts via its binding to the aromatic hydrocarbon (Ah) receptor. We therefore examined the ability of HCB to interact with this receptor in vitro and in vivo. HCB, at concentrations of 1 microM or higher, inhibited the specific binding of [3H]TCDD (0.3 nM) to the Ah receptor in vitro, whereas the solubility of [3H]TCDD was affected only at 100 microM HCB. The inhibition was competitive, with a KI of approximately 2.1 microM. In rats fed a diet containing 3000 ppm HCB for varying times (4 h to 7 days), the specific binding of [3H]TCDD in hepatic cytosol was reduced by up to 40%, as observed previously for known Ah receptor agonists. The decrease in [3H]TCDD specific binding in cytosol of HCB-treated rats was due principally to a decrease in the number of binding sites for [3H]TCDD rather than competition from residual HCB. As shown by immunoblotting and radioimmunoassay, HCB induced the cytochrome P450 isozymes P450c and P450d, which are regulated by the Ah receptor, as well as the phenobarbital-inducible isozymes P450b and P450e. Together these results indicate that HCB is a weak agonist for the Ah receptor, and suggest that some of its effects may be mediated by its interaction with this gene-regulatory protein.

Induction of porphyria in the rat by chronic versus acute exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin

Chronic oral administration of 1 microgram . kg-1 . week-1 of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to female rats for 16 weeks resulted in hepatic porphyria. In contrast, administration of single oral doses as high as 30 micrograms/kg did not produce porphyria, either acutely or 16 weeks later. Activities of hepatic drug-metabolizing enzymes [aryl hydrocarbon hydroxylase (AHH) and glucuronyl transferase] were increased by chronic oral doses of TCDD as low as 0.01 microgram . kg-1 . week-1. When animals were dosed with TCDD chronically and then allowed to recover for 6 months, AHH and glucuronyl transferase activities returned toward normal (98 and 86% recovery). However, animals showed only partial recovery from TCDD-induced porphyria. Hepatic porphyrin levels did decrease during this period, but urinary porphyrins and the rate-limiting enzyme in porphyrin synthesis, delta-aminolevulinic acid synthetase, remained maximally elevated during the 6-month recovery period. It is concluded that single doses of TCDD do not produce porphyria in the rat, but that TCDD is porphyrogenic when given chronically. Moreover, when TCDD administration is stopped, recovery from the porphyrogenic effects of TCDD is very slow and does not correlate with the biological half-life of TCDD in the rat.

Induction of specific cytochrome P-450 isozymes by methylenedioxyphenyl compounds and antagonism by 3-methylcholanthrene

Two methylenedioxyphenyl compounds, isosafrole (5-propenyl-1,3-benzodioxole) and an analog, 5-t-butyl-1,3-benzodioxole (BD), differ markedly as inducers of cytochrome P-450 isozymes in rat liver microsomes. Isosafrole is a mixed-type inducer, inducing P-450b, P-450c, and P-450d. In contrast, BD is a phenobarbital-type inducer, increasing P-450b, but producing little or no increase in P-450c or P-450d. Similarly, isosafrole increases the amount of translatable mRNA for P-450b, c and d, while BD induces only the mRNA for P-450b. Dimethylation of the methylene bridge carbon of BD to give 2,2-dimethyl-5-t-butyl-1,3-benzodioxole (DBD) blocks the formation of NADPH-reduced Type III metabolite-P-450 complexes in vitro, and diminishes but does not abolish the ability of the compound to induce P-450b. Western blots of microsomes from isosafrole and BD-treated rat livers confirm that in contrast to isosafrole, BD does not induce P-450d or P-450c. However, the antibody to P-450d recognizes two new polypeptides (approximately 50K Mr) from sodium dodecyl sulfate-polyacrylamide gels of liver microsomes from BD-treated rats. These polypeptides are not observed in control, isosafrole, 3-methylcholanthrene (3-MC), or DBD-treated rats. They are intensified by coadministration of 3-MC with BD and may represent either modified isozyme-metabolite adducts or degradation products of P-450d. However, the polypeptides could not be generated in vitro by addition of BD to 3-MC-induced microsomes with NADPH under conditions which produced spectral metabolite complexes, or in a reconstituted system with P-450d. The two methylenedioxyphenyl compounds do not form stable metabolite complexes with the same P-450 isozymes. BD formed distinct spectral metabolite complexes in vitro with both P-450b and P-450c but not with P-450d in a reconstituted system. In contrast, isosafrole forms metabolite complexes with all three isozymes. Coadministration of 3-MC with BD blocked induction of P-450b by 80% and produced a similar repression of its translatable mRNA. This finding indicates that 3-MC type inducers not only induce certain cytochrome P-450 isozymes, but also repress synthesis of other isozymes.

Effects of dose and route of exposure on dioxin disposition

Abstract The absorption, distribution, metabolism and excretion of 2,3,7,8-tetrabromodibenzo- p -dioxin (TBDD) was studied in male F344 rats. Oral absorption was dose-dependent. Absorption of 1 nmol/kg by both the oral and intratracheal (itr) routes was 〉80%, whereas only 〉12% was absorbed through the skin. Tissue distribution was dependent on dose, time, and route of exposure. Liver:fat ratios of 3.4, 2.9, 2.0, and 1.5 were observed 3 days after iv, oral, itr, and dermal administration, respectively, of 1 nmol/kg. Liver:fat ratios were 0.2 and 2.6 by 56 days after 1 and 100 nmol/kg iv, respectively. Dose-dependent elimination in urine and feces was observed beginning 3 weeks after iv administration. However, auto-induction of dioxin metabolism did not occur in vivo when evaluated by biliary excretion of [ 3 H]TBDD or [ 3 H]TCDD in pretreated and naive rats. Dose-response profiles for TBDD induction of hepatic cytochromes CYP1A1 and CYP1A2 indicated the latter to be a more sensitive response. Finally, comparison of the dose-response behavior for TBDD induction of hepatic CYP1A2 with hepatic concentrations of TBDD suggests that induction of CYP1A2 alone does not account for nonlinearities in dioxin disposition exemplified by dose-related increases in the ratio of dioxin concentrations in liver and fat.

Effect of hexachlorobenzene on the specific binding of 2,3,7,8,-TCDD to the hepatic Ah receptor

The ability of HCB to interact with the Ah receptor was investigated in vitro and in vivo. HCB, up to 1.0 μM, was not a potent in vitro competitor for the specific binding of [3H]-TCDD (0.3 nM) to rat hepatic cytosol. Administration of HCB (3000 ppm in the diet) to rats for up to 7 days resulted in a decrease in the specific binding of [3H]-TCDD to hepatic cytosol, as compared to pair-fed control rats. These results suggest that HCB may be able to interact, either directly or indirectly, with the hepatic Ah receptor in vivo.