Stereoselective metabolism of the (+)-(S,S)- and (-)-(R,R)-enantiomers of trans-3,4-dihydroxy-3,4-dihydrobenzo[c]-phenanthrene by rat and mouse liver microsomes and by a purified and reconstituted cytochrome P-450 system

Metabolism of (+)-, (-)-, and (+/-)-trans-3,4-dihydroxy-3, 4-dihydrobenzo[c]phenanthrenes by liver microsomes from rats and mice and by a purified monooxygenase system reconstituted with cytochrome P-450c has been examined. Bay-region 3,4-diol 1,2-epoxides are minor metabolites of both enantiomers of the 3,4-dihydrodiol with liver microsomes from 3-methylcholanthrene-treated rats or with the reconstituted system (less than 10% of total metabolites). Microsomes from control and phenobarbital-treated rats and from control mice form higher percentages of these diol epoxides (13-36% of total metabolites). Microsomes from 3-methylcholanthrene-treated rats and cytochrome P-450c in the reconstituted system form exclusively the diol expoxide-1 diastereomer, in which the benzylic hydroxyl group and oxirane oxygen are cis to each other, from the (+)-(3S,4S)-dihydrodiol. The same enzymes selectively form the diol expoxide-2 diastereomer, with its oxirane oxygen and benzylic hydroxyl groups trans to each other, from the (-)-(3R,4R)-dihydrodiol (77% of the total diol epoxides). Liver microsomes from control rats show similar stereoselectivity whereas liver microsomes from phenobarbital-treated rats and from control mice are less stereoselective. Three bis-dihydrodiols and three phenolic dihydrodiols are also formed from the enantiomeric 3,4-dihydrodiols of benzo[c]phenanthrene. A single diastereomer of one of these bis-dihydrodiols with the newly introduced dihydrodiol group at the 7,8-position accounts for 79-88% of the total metabolites of the (-)-(3R,4R)-dihydrodiol formed by liver microsomes from 3-methylcholanthrene-treated rats or by the reconstituted system containing epoxide hydrolase. In contrast, the (+)-(3S,4S)-dihydrodiol is metabolized to two diastereomers of this bis-dihydrodiol, a third bis-dihydrodiol, and two phenolic dihydrodiols.     

Induction, activation and inhibition of epoxide hydrase: an anomalous prevention of chlorobenzene-induced hepatotoxicity by an inhibitor of epoxide hydrase

Epoxide hydrase activity, measured with [³H]styrene oxide as substrate, is present in mammalian liver, kidney, lung, intestine and skin. The hepatic level of the enzyme, measured in vitro with [³H]styrene oxide, benzene oxide or naphthalene-1,2-oxide, is elevated substantially by pretreatment of rats with phenobarbital and to a lesser extent by pretreatment with 3-methylcholanthrene. Metyrapone and 1-(2-isopropylphenyl)-imidazole, two monooxygenase inhibitors, activate epoxide hydrase in vitro, but have no demonstrable effect on the enzyme in vivo. 3,3,3-Trichloropropene oxide, a potent in vitro inhibitor of epoxide hydrase, has no effect on monooxygenase activity measured in vitro with [³H]benzenesulfonanilide. Trichloropropene oxide is extremely toxic. In sub-lethal dosages, it does not significantly inhibit epoxide hydrase activity in vivo, although it and several other epoxides do react with and thereby reduce hepatic levels of glutathione. Cyclohexane oxide, another potent in vitro inhibitor of epoxide hydrase, reduces hepatic glutathione levels to 10% of control values. This relatively non-toxic substance should potentiate the hepatotoxicity of chlorobenzene by inhibiting further metabolism of the toxic chlorobenzene oxide intermediate through either hydration or conjugation with glutathione. Instead, co-administration of cyclohexene oxide and chlorobenzene significantly reduces the rate of metabolism of [¹⁴C]chlorobenzene and prevents the hepatic centrilobular necrosis caused by chlorobenzene in rats. Arene oxide-mediated hepatotoxicity apparently is dependent upon a variety of factors including both rates of formation and degradation of arene oxides in tissue. The presently known hydrase inhibitors are not sufficiently selective in their effects on liver cells to permit a quantitative assessment of the relative importance of these factors.     

Metabolism and hepatotoxicity of the naturally occurring benzo[b]pyran precocene I

The in vitro metabolism of precocene I by liver microsomes from control and treated rats and the effects of precocene I on the function and histology of the rat liver were examined. The major metabolites (80-90% of total metabolites) from all microsomal preparations were the cis and trans 3,4-diols of precocene I produced with a cis/trans isomer ratio of 1:2. These diols appear to arise mainly by spontaneous hydrolysis of precocene I 3,4-oxide. (+)-(3R,4R)-cis- and (-)-(3R,4S)-trans-precocene I 3,4-diols were the predominant enantiomers of the 3,4-diol formed. The enantiomeric excess of these diols (2-50%) is dependent on the microsomal preparation, with microsomes from control rats exhibiting the highest stereoselectivity and microsomes from phenobarbital-treated rats the least. 6-Hydroxyprecocene I was the next major metabolite and was formed to the extent of 5% (control), 10% and 17% (phenobarbital and 3-methylcholanthrene treatment, respectively) of total metabolites. Treatment of rats with a single i.p. dose of precocene I (300 mg/kg) resulted in extensive hepatic damage as evidenced by a marked increase of plasma glutamic pyruvic transaminase levels and histologic observation in liver sections of severe centrolobular necrosis. Although phenobarbital treatment of rats increased the rate of liver microsomal metabolism of precocene I by approximately 50% (nmol products/nmol cytochrome P-450/min) compared to liver microsomes from control rats, hepatic damage caused by precocene I was not significantly affected. Depletion of glutathione levels in the rats with diethyl maleate prior to precocene I treatment dramatically increased the severity of hepatic insult, whereas treatment of the rats with the mixed function oxidase inhibitor piperonyl butoxide prior to treatment with precocene I blocked hepatic damage. Treatment of rats with cysteamine prior to treatment with precocene I protected the animals against the toxic effects. Neither cis nor trans precocene I 3,4-diol nor 3,4-dihydroprecocene I elicited impaired liver function or cellular damage. The above results are consistent with the view that precocene I 3,4-oxide is the metabolite responsible for the hepatotoxic effects observed when precocene I is injected into rats.     

Novel stereoselectivity of rat liver cytochrome(s) P450 toward enantiomers of the trans-1,2-dihydrodiol of triphenylene

Metabolism of 3H-labeled (+)-(S,S)- and (-)-(R,R)-1,2-dihydrodiols of triphenylene by rat liver microsomes and 11 purified isozymes of cytochrome P450 in a reconstituted monooxygenase system has been examined. Although both enantiomers were metabolized at comparable rates, the distribution of metabolites between phenolic dihydrodiols and bay-region, 1,2-diol 3,4-epoxide diastereomers varied substantially with the different systems. Treatment of rats with phenobarbital (PB) or 3-methylcholanthrene (MC) caused a slight reduction or less than a twofold increase, respectively, in the rate of total metabolism (per nanomole of cytochrome P450) of the enantiomeric dihydrodiols compared to microsomes from control rats. Among the 11 isozymes of cytochrome P450 tested, only cytochromes P450c (P450IA1) and P450d (P450IA2) had significant catalytic activity. With either enantiomer of triphenylene 1,2-dihydrodiol, both purified cytochrome P450c (P450IA1) and liver microsomes from MC-treated rats formed diol epoxides and phenolic dihydrodiols in approximately equal amounts. Purifed cytochrome P450d (P450IA2), however, formed bay-region diol epoxides and phenolic dihydrodiols in an 80:20 ratio. Interestingly, liver microsomes from control or PB-treated rats produced only diol epoxides and little or no phenolic dihydrodiols. The diol epoxide diastereomers differ in that the epoxide oxygen is either cis (diol epoxide-1) or trans (diol epoxide-2) to the benzylic 1-hydroxyl group. With either purified cytochromes P450 (isozymes c or d) or liver microsomes from MC-treated rats, diol epoxide-2 is favored over diol epoxide-1 by at least 4:1 when the (-)-enantiomer is the substrate, while diol epoxide-1 is favored by at least 5:1 when the (+)- enantiomer is the substrate. In contrast, with liver microsomes from control or PB-treated rats, formation of diol epoxide-1 relative to diol epoxide-2 was favored by at least 2:1 regardless of the substrate enantiomer metabolized. This is the first instance where the ratio of diol epoxide-1/diol epoxide-2 metabolites is independent of the dihydrodiol enantiomer metabolized. Experiments with antibodies indicate that a large percentage of the metabolism by microsomes from control and PB-treated rats is catalyzed by cytochrome P450p (P450IIIA1), resulting in the altered stereoselectivity of these microsomes compared to that of the liver microsomes from MC-treated rats.     

Stereoselectivity of rat liver cytochrome P-450c on formation of benzo[a]pyrene 4,5-oxide

N-Nitroethylenediamine is a mushroom product which closely resembles the neurotransmitter 4-aminobutyrate, GABA. The nitramine is sequentially accepted as a substrate by the GABA-catabolizing enzymes GABA aminotransferase (EC 2.6.1.19) and succinic semialdehyde dehydrogenase (EC 1.2.1.16). In view of the steric and ionic similarity of the nitramino group to the carboxymethyl group, nitramines may prove generally useful for enzymological and pharmacological purposes as analogs of carboxylic acids.     

Inhibition of Werner syndrome helicase activity by benzo[a]pyrene diol epoxide adducts can be overcome by replication protein A

RecQ helicases are believed to function in repairing replication forks stalled by DNA damage and may also play a role in the intra-S-phase checkpoint, which delays the replication of damaged DNA, thus permitting repair to occur. Since little is known regarding the effects of DNA damage on RecQ helicases, and because the replication and recombination defects in Werner syndrome cells may reflect abnormal processing of damaged DNA associated with the replication fork, we examined the effects of specific bulky, covalent adducts at N(6) of deoxyadenosine (dA) or N(2) of deoxyguanosine (dG) on Werner (WRN) syndrome helicase activity. The adducts are derived from the optically active 7,8-diol 9,10-epoxide (DE) metabolites of the carcinogen benzo[a]pyrene (BaP). The results demonstrate that WRN helicase activity is inhibited in a strand-specific manner by BaP DE-dG adducts only when on the translocating strand. These adducts either occupy the minor groove without significant perturbation of DNA structure (trans adducts) or cause base displacement at the adduct site (cis adducts). In contrast, helicase activity is only mildly affected by intercalating BaP DE-dA adducts that locally perturb DNA double helical structure. This differs from our previous observation that intercalating dA adducts derived from benzo[c]phenanthrene (BcPh) DEs inhibit WRN activity in a strand- and stereospecific manner. Partial unwinding of the DNA helix at BaP DE-dA adduct sites may make such adducted DNAs more susceptible to the action of helicase than DNA containing the corresponding BcPh DE-dA adducts, which cause little or no destabilization of duplex DNA. The single-stranded DNA binding protein RPA, an auxiliary factor for WRN helicase, enabled the DNA unwinding enzyme to overcome inhibition by either the trans-R or cis-R BaP DE-dG adduct, suggesting that WRN and RPA may function together to unwind duplex DNA harboring specific covalent adducts that otherwise block WRN helicase acting alone.     

Effect of DNA modifications on DNA processing by HIV-1 integrase and inhibitor binding: role of DNA backbone flexibility and an open catalytic site

Integration of the viral cDNA into host chromosomes is required for viral replication. Human immunodeficiency virus integrase catalyzes two sequential reactions, 3'-processing (3'-P) and strand transfer (ST). The first integrase inhibitors are undergoing clinical trial, but interactions of inhibitors with integrase and DNA are not well understood in the absence of a co-crystal structure. To increase our understanding of integrase interactions with DNA, we examined integrase catalysis with oligonucleotides containing DNA backbone, base, and groove modifications placed at unique positions surrounding the 3'-processing site. 3'-Processing was blocked with substrates containing constrained sugars and alpha-anomeric residues, suggesting that integrase requires flexibility of the phosphodiester backbone at the 3'-P site. Of several benzo[a]pyrene 7,8-diol 9,10-epoxide (BaP DE) adducts tested, only the adduct in the minor groove at the 3'-P site inhibited 3'-P, suggesting the importance of the minor groove contacts for 3'-P. ST occurred in the presence of bulky BaP DE DNA adducts attached to the end of the viral DNA suggesting opening of the active site for ST. Position-specific effects of these BaP DE DNA adducts were found for inhibition of integrase by diketo acids. Together, these results demonstrate the importance of DNA structure and specific contacts with the viral DNA processing site for inhibition by integrase inhibitors.     

In vivo aging characteristics of silicone gel breast implants compared to lot-matched controls

A study was conducted to investigate the effect of in vivo aging on the physical, mechanical, and chemical properties of Silastic II gel-filled breast implants. In the study, the properties of 16 Silastic II gel-filled explants (retrieved from eight patients), with in vivo duration times ranging from 4 months to 13 years, were compared with lot-matched control (unimplanted) samples. Tensile and tear strength properties were measured for both explant and control shells by using identical testing protocols. The tensile strength properties of shells, which were extracted with hexane to remove non-cross-linked silicones, were also measured. Swelling measurements were used to determine the average molecular weight between cross-links (or entanglements). In addition, scanning electron microscopy was applied in the comparison of the morphological features of the explants and their lot-matched controls. The results of the study suggest that the silicone polymer used to fabricate the shells does not undergo appreciable degradation for up to 13 years in vivo. The study represents an investigation of the world's largest known inventory of explanted breast implants with lot-matched controls.     

Scanning electron microscopy characterization of surgical instrument damage to breast implants.

In this article, mechanisms of breast-implant failure caused by surgical instruments commonly used to perform implantation, breast biopsies, needle localization procedures, cyst aspirations, and explantation are described. Failure was artificially induced in breast-implant shells using various types of surgical instruments, including scalpels, suture needles, hypodermic needles, hemostats, and Adson forceps. Field-emission scanning electron microscopy (SEM) was used to document the morphology of the failure sites produced by these instruments. Micrographs were used to categorize failure according to a specific type of surgical instrument. SEM micrographs were also obtained on explants that failed in situ, and the morphology of the corresponding failure sites was examined. The study was designed to document a range of failure mechanisms associated with gel-filled, saline-filled, double-lumen (saline-gel), and soybean oil-filled implants. The results of the study also demonstrate that SEM can often be used to determine the cause of breast-implant failure.