Dihalomethanes can produce liver tumors in mice but not in rats, and concern exists about the risk of these compounds to humans. Glutathione (GSH) conjugation of dihalomethanes has been considered to be a crifical event in the bioactivation process, and risk assessment is based upon this premise; however, there is little experimental support for this view or information about the basis of genotoxicity. A plasmid vector containing rat GSH S-transferase 5-5 was transfected into the SalmoneUla typhimurium tester strain TA1535, which then produced active enzyme. The transfected bacteria produced base-pair revertants in the presence ofethylene dihalides or dihalomethanes, in the order CH2Br2 > CH2BrCl > CH2CI2. However, revertants were not seen when cells were exposed to GSH, CH2Br2, and an amount of purified GSH S-ransferase 5-5 (20-fold excess in amount of that expressed within the cells). HCHO, which is an end product of the reaction of GSH with dihalomethanes, also did not produce mutations. S-(1-Acetoxymethyl)GSH was prepared as an analog of the putative S-(1-halomethyl)GSH reactive intermediates. This analog did not produce revertants, consistent with the view that activation of dihalomethanes must occur within the bacteria to cause genetic damage, presenting a model to be considered in studies with mammalian cells. S-(1-Acetoxymethyl)GSH reacted with 2'-deoxyguanosine to yield a major adduct, identified as S-[l-(N2-deoxyguanosinyl)methyl]GSH.
Aromatic amines are bioactivated to electrophilic compounds that react with DNA, predominantly at the Ca position of guani bases. This site is weakly nudeephilic and it has been proposed that the Cs adduct Is the final product after initial N7-adduct formation. To consider this possibility, we reacted several C-substituted nine derivatives with N-acetoxy-2-aminofluorene, prepared in situ from 2-acetylsalicylic acid and N-hydroxy-2-aminofluorene. With C5,N'-dimethylguanine, an adduct was isolated in good yield that was consistent, by NMR and mass spectral characterization, with a structure involving cardnogen substitution at the N7 position of guanine and linked through the 2-aminofluorenyl nitrogen-N-(C6,N9-dimethylguanin-N7-yl)-2-aminofluorene. This adduct could be easily reduced with NaBH4, consistent with the proposed N7-adduct structre. The same reaction was also carried out with Cs-methylguanoslne and Cs-methyldeoxyguanosine and similar adducts were isolated. In contrast, C0-bromoguanosine reacted with N-acetoxy-2-ainlrene to yield the Cs-substituted arylamine adduct N-(guanosin-CO-yl)-2-aminofluorene directly. These products are uniquely consistent with a scheme in which Cs-adduct formation is preceded by an initial electrophilic substitution on the N7 atom, which is postulated to be a general reaction for activated arylamines and heterocyclic amines.Arylamines and heterocyclic amines constitute an extremely important class of chemical carcinogens (1, 2). They are bioactivated by oxidative and conjugative metabolism and the ultimate carcinogen is thought to be either the N-hydroxyarylamine or an O-esterified derivative formed by enzymatic conjugation (3) (Fig. 1). These compounds are strongly electrophilic and react through an SN2 mechanism with cellular DNA to yield adducted bases (4). The major adduct formed is generally at the C8 position of guanine residues (5). The C8 position is a favored site for certain radical addition reactions but seems weakly nucleophilic and does not react readily with other electrophilic species (6, 7). Indeed, most ofthe known guanyl-C8-substituted adducts are formed with radicals and arylamines (6), with a few exceptions that could proceed through alternative mechanisms but yield products that are not consistent with simple nucleophilic attack by the C8 atom [e.g., adducts derived from bifunctional reagents (8)]. This apparent contradiction has led to several proposals for alternative pathways for C8-adduct formation employing some type of intermediate site of adduction (9, 10). Because of the greater nucleophilicity and close proximity of the N7 atom, this would seem to be the most favored site for such an intermediate; i.e., 2. There have been several lines of evidence indirectly supporting such a view. Tarpley et al. (10) showed that there was rapid depurination upon treatment of DNA with activated arylamines, and Kohda et aL (11) showed that there was an increase in the amount of 7,8-dihydro-8-oxoguanine formed after reaction of DNA with activated 4-aminoquinol...
Cytochrome P450 3A4 (CYP3A4), an enzyme that is highly expressed in the human liver and small intestine, plays a major role in the metabolism of a large variety of xenobiotics, including an estimated 50% of therapeutic drugs, as well as many endogenous compounds. The expression of CYP3A4 can be induced by xenobiotics. Such induction leads to accelerated metabolism of the xenobiotics themselves (autoinduction) or of concomitantly administered CYP3A4 substrates/drugs, thereby significantly altering their pharmacokinetic and pharmacodynamic profiles. During the past decade, much progress has been made in our understanding of the biological mechanisms responsible for regulation of CYP3A4 expression. It is now known that many xenobiotics induce CYP3A4 expression via the pregnane X receptor (PXR) pathway, while others are thought to act through the constitutive androstane receptor (CAR) and the vitamin D receptor (VDR). As a result, most pharmaceutical companies have recognized that it is important to evaluate CYP3A4 induction potential preclinically and are using primary cultures of human hepatocytes and/or PXR reporter gene assays. In general, the results from these two assay methods correlate well. The reporter gene assays in particular can be used to rapidly screen hundreds of drug candidates, whereas methods using primary human hepatocyte cultures may more accurately assess the potential for CYP3A4 induction in vivo. Although it is important to consider CYP3A4 induction in the early stages of the drug development process, it should be recognized that the assessment of induction potential preclinically is a difficult and imprecise endeavor and can be complicated by many factors.
The mutagenicity of 1,2-dibromoethane is highly dependent upon its conjugation to glutathione by the enzyme glutathione S-transferase. The conjugates thus formed can react with DNA and yield almost exclusively N7-guanyl adducts. We have synthesized the S-haloethyl conjugates of cysteine and glutathione, as well as selected methyl ester and N-acetyl derivatives, and compared them for ability to produce N7-guanyl adducts with calf thymus DNA. The cysteine compounds were found to be more reactive toward calf thymus DNA and yielded higher adduct levels than did the glutathione compounds. Adduct levels tended to be suppressed when there was a net charge on the compound and were not affected by substitution of bromine for chlorine, as expected for a mechanism known to involve an intermediate episulfonium ion. Sequence-selective alkylation of fragments of pBR322 DNA was investigated. The compounds produced qualitatively similar patterns of alkylation, with higher levels of alkylation at runs of guanines. The compounds were also tested for their ability to act as direct mutagens in Salmonella typhimurium TA98 and TA100. None of the compounds caused mutations in the TA98 frameshift mutagenesis assay. In the strain TA100, where mutation of a specific guanine by base-pair substitution produces reversion, all compounds were found to produce mutations, but the levels of mutagenicity did not correlate at all with the levels of DNA alkylation. The ratio of mutations to adducts varied at least 14-fold among the various N7-guanyl adducts examined.(ABSTRACT TRUNCATED AT 250 WORDS)
It has been proposed that aluminum ion is a contributing factor in a variety of neurological diseases. In many of these diseases, aberrations in the cytoskeleton have been noted. The effects of aluminum ion on the in vitro assembly of tubulin into microtubules has been examined by determining the association constants for the metal ion-guanosine triphosphate-tubulin ternary complex required for polymerization. The association constant for aluminum ion was approximately 10(7) times that of magnesium ion, the physiological mediator of microtubule assembly. In addition, aluminum ion at 4.0 X 10(-10) mole per liter competed effectively with magnesium ion for support of tubulin polymerization when magnesium ion falls below 1.0 millimole per liter. The microtubules produced by aluminum ion were indistinguishable from those produced by magnesium ion when viewed by electron microscopy, and they showed identical critical tubulin concentrations for assembly and sensitivities to cold-induced depolymerization. However, the rate of guanosine triphosphate hydrolysis and the sensitivity to calcium ion-induced depolymerization, critical regulatory processes of microtubules in vivo, were markedly lower for aluminum ion microtubules than for magnesium ion microtubules.
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