Step In Metabolic Activation Research Articles

The Role of Intramolecular Reactions and Chemical Degradation in the Apparent Biotransformation Pathways of a Series of SYK Inhibitors.

In vitro metabolism studies of the spleen tyrosine kinase inhibitors AZ-A and AZ-B identified four unusual metabolites. M1 (mass-to-charge ratio 411) was formed by both molecules and was common to several analogs (AZ-C to AZ-H) sharing the same core structure, appearing to derive from the complete loss of a pendent 3,4-diaminotetrahydropyran ring and pyrazole ring cleavage resulting in a nonobvious metabolite. M2-M4 were formed by AZ-A and a subset of the other compounds only and apparently resulted from a sequential loss of H2 from parent. Initial attempts to isolate M3 for identification were unsuccessful due to sample degradation, and it was subsequently found that M2 and M3 underwent sequential chemical degradation steps to M4. M4 was successfully isolated and shown by mass spectrometry and NMR spectroscopy to be a tricyclic species incorporating the pyrazole and the 3,4-diaminotetrahydropyran groups. We propose that this arises from an intramolecular reaction between the primary amine on the tetrahydropyran and a putative epoxide intermediate on the adjacent pyrazole ring, evidence for which was generated in a β-mercaptoethanol-trapping experiment. The loss of the tetrahydropyran moiety observed in M1 was found to be enhanced in an analog that was unable to undergo the intramolecular reaction step, leading us to propose two possible reaction pathways originating from the reactive intermediate. Ultimately, we conclude that the apparently complex and unusual metabolism of this series of compounds likely resulted from a single metabolic activation step forming an epoxide intermediate, which subsequently underwent intramolecular rearrangement and/or chemical degradation to form the final observed products. SIGNIFICANCE STATEMENT: The current work provides an unusual biotransformation example showing the potential for intramolecular reactions and chemical degradation to give the appearance of complex metabolism arising from a single primary route of metabolism.

Chemistry, Cyclophosphamide, Cancer Chemotherapy, and Serendipity: Sixty Years On

Cambridge Dictionary: serendipity | noun | the phenomenon of finding interesting or valuable things by chance.
 The year 2019 marked the 60th anniversary of the approval of cyclophosphamide (CP) as an anticancer by the U.S. Food & Drug Administration in 1959 for the treatment of lymphoma. Between 1959 and 2019 there were ~50,000 publications listed in PubMed that have CP in the title and/or abstract, with these annual numbers showing a continual increase, and over 1,800 such articles in 2019 alone. The discovery of CP is a prime example of serendipity in science, which also applies to key elements of the metabolism and pharmacological basis for the specificity of the cytotoxicity of CP toward cancer cells. Phosphoramide mustard (PM), HO(H2N)P(O)N(CH2CH2Cl)2, the principal metabolite of CP with DNA alkylating activity, was synthesized and reported by Friedman and Seligman in 1954 prior to the discovery of CP. Interestingly, the original drug design premise for synthesizing PM, which was based on elevated phosphamidase enzyme activity in cancer cells proved to be incorrect. While this wrong premise also led to the synthesis of CP, as a six-membered ring cyclic phosphamidase-activated precursor of PM, the actual metabolic conversion of CP to PM was subsequently found to involve a surprisingly complex array of metabolites and metabolic pathways, all completely unrelated to phosphamidase. Although the molecular structure of CP has an asymmetrically substituted, i.e. chiral phosphorus center, the racemic mixture of the Rp and Sp enantiomers of CP was used throughout its initial investigations and subsequent clinical trials despite the involvement of an initial enzyme-mediated metabolic activation step, which could, in principle, be stereoselective for only one of the enantiomers of CP. Stereochemical investigations along those lines were eventually carried out, but the results did not warrant replacement of racemic CP with either enantiomer in the clinic. Amazingly, there are now ~4,000 structural congeners of PM listed Chemical Abstracts, but none have led to an anticancer drug superior to CP. This account provides a synopsis of the key chemistry and stereochemistry investigations that comprise this story of CP, as a remarkable instance of serendipity in science, and my chance involvement in the unfolding of this fascinating story.

Case Study 12: Roadmap to Quantifying Ago2-Mediated siRNA Metabolic Activation Kinetics.

Therapeutic siRNA is a prodrug that requires Ago2-mediated site-specific hydrolysis of the sense strand before RNA interference can occur. Although this metabolic activation step was first described 15years ago, the kinetics of this reaction, and its relationship to in vivo siRNA efficacy, remains unexplored in the literature. To provide a roadmap to address these gaps, we describe a liquid chromatography-mass spectrometry method to monitor formation of the cleaved sense-strand metabolites in a reconstituted system. In the absence of metabolite standards for quantitation, we apply an ionization efficiency correction across a panel of siRNA molecules and find that it improves in vitro-in vivo correlation in a transgenic mouse model. Finally, we lay out a case for why Michaelis-Menten kinetics will likely be inadequate for describing Ago2-mediated metabolic activation kinetics, and propose several alternative models that can be solved numerically and applied to quantitated kinetic data when it becomes available.

Screening of molecular cell targets for carcinogenic heterocyclic aromatic amines by using CALUX® reporter gene assays.

Heterocyclic aromatic amines (HCAs) are compounds formed when meat or fish are cooked at high temperatures for a long time or over an open fire. To determine which pathways of toxicity are activated by HCAs, nine out of the ten HCAs known to be carcinogenic in rodents (2-amino-9H-pyrido[2,3-b]indole (AαC), 2-aminodipyrido[1,2-a:3',2-d]imidazole (Glu-P-2), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2)) were tested in the estrogen receptor α (ERα), androgen receptor (AR), glucocorticoid receptor (GR), peroxisome proliferator-activated receptor γ2 (PPARγ2), polycyclic aromatic hydrocarbons (PAH), Nrf2, and p53 CALUX® reporter gene assays. Trp-P-1 was the only HCA that led to a positive response in the ERα, PPARγ2, and Nrf2 CALUX® assays. In the PAH CALUX® assay, Trp-P-2, MeAαC, and AαC induced luciferase activity to a greater extent than MeIQ and PhIP. In the p53 CALUX® assay without a coupled metabolic activation, only Trp-P-1 and Trp-P-2 enhanced luciferase expression; when a metabolic activation step was coupled to the p53 CALUX® assay, Trp-P-1, Glu-P-2, MeIQ, MeIQx, and PhIP induced a positive response. No HCA was positive in the AR and GR CALUX® assays. Taken together, the results obtained show that the battery of CALUX® assays performed in the present study can successfully be used to screen for molecular cell targets of carcinogenic compounds such as HCAs.

Beyond monotherapy: Integrating immunotherapy into current treatment regimens

While we have shown that these urinary metabolites are risk biomarkers for lung cancer in these studies, they are not fully predictive. This is because these biomarkers are mainly measuring exposure but do not take into account the critical metabolic activation step leading to the formation of DNA adducts which cause the multiple mutations observed in lung cancer. Quantitation of DNA adducts is challenging because of their low levels, typically less than 1 adduct per 10 normal bases, and the small amounts of DNA available for studies in living humans. To address this question, we are developing high resolution mass spectrometric methods for quantifying oral cell DNA adducts as a surrogate for DNA adduct formation in the lung. In one recent study, we found remarkably high levels of DNA adducts of tobaccospecific compounds in in the oral mucosa cells of smokers compared to non-smokers. We are also able to quantify formaldehyde-DNA adducts in oral mucosa cells. As mass spectrometric methods for analysis of carcinogen-DNA adducts become increasingly more sensitive and specific, it appears likely that quantitation of a panel of tobacco carcinogen DNA adducts in smokers, leading to identification of susceptible individuals, may be feasible.

Synthesis and Antimycobacterial Activity of Symmetric Thiocarbohydrazone Derivatives against Mycobacterium bovis BCG.

In this work, we reported the synthesis and evaluation of antimycobacterial and antifungal activity of a series of thiocarbohydrazone derivatives which are thiacetazone congeners. The target compounds were synthesized in superior yields by reacting thiocarbohydrazide with different aromatic aldehydes and methyl ketones. Compounds 8, 19 and 25 were found to be the most potent derivatives, exhibiting acceptable activity against Mycobacterium bovis BCG compared to thiacetazone and ethambutol as reference substances. Compounds 8, 15 and 25 exhibited the highest activity against Candida albicans. The most active compounds had a completely different aromatic ring system with various electronic, steric and lipophilic natures. This is understandable in light of the fact that carbohydrazone derivatives must undergo a metabolic activation step before exerting their anti-TB activity and different SAR rules govern each one of these two processes.

FORMATION OF BENZYLIC ALCOHOLS AND MESO-ALDEHYDES BY ONE-ELECTRON OXIDATION OF DMBA, A MODEL FOR THE FIRST METABOLIC STEP IN METHYLATED CARCINOGENIC HYDROCARBON ACTIVATION

The hypothesis that hydroxylation of the 7-methyl group is the first step in metabolic activation of 7,12-dimethylbenz[a]anthracene (DMBA) was advanced over three decades ago (1, 2). A considerable body of evidence supports the hypothesis (3). A chemical model for the oxidative metabolism of DMBA may be useful in understanding the mechanism of metabolic activation of DMBA and other methylated carcinogenic hydrocarbons, particularly the first step. Here we show that a nonenzymatic one-electron oxidation pathway transforms DMBA to carcinogenic alcohol and meso-aldehyde metabolites. The results are consistent with the hypothesis that hydroxylation of the 7-methyl and/or 12-methyl groups is the first metabolic step in DMBA activation. The hypothesis predicts that hydroxylation of other meso-methyl-substituted hydrocarbons is the first essential step in the metabolic activation of methylated carcinogenic hydrocarbons.

Approaches to cancer prevention based on an understanding of N-nitrosamine carcinogenesis.

Understanding carcinogenesis is critical for development of rational approaches to cancer prevention. This paper uses N-nitrosamine carcinogenesis as an example. N-Nitrosamines are a large group of potent carcinogens. Approximately 300 different N-nitrosamines are carcinogenic. At least 30 animal species are responsive to their effects. There is little doubt that humans exposed to sufficient amounts of N-nitrosamines would also be susceptible to their carcinogenic effects. Human exposure to preformed N-nitrosamines occurs through the diet, in certain occupational settings, and through the use of tobacco products, cosmetics, pharmaceutical products, and agricultural chemicals. Diminishing human exposure to these carcinogens is one approach to prevention of cancer, and this has been accomplished in many instances, although exposure to N-nitrosamines in tobacco products is still unacceptably high. Human exposure to N-nitrosamines also occurs by nitrosation of amines in the body, via their acid or bacterial catalyzed reaction with nitrite, or by reaction with products of nitric oxide generated during inflammation or infection. A second approach toward prevention of N-nitrosamine carcinogenesis is inhibition of this endogenous N-nitrosamine formation. Substantial reductions have been achieved with ascorbic acid and other nitrite scavengers. N-Nitrosamines undergo a simple cytochrome P450-mediated metabolic activation step, which is critical for their carcinogenicity. The third approach involves the use of chemopreventive agents that block this step, or other steps in the carcinogenic process. A large number of potent chemopreventive agents against nitrosamine carcinogenesis have been identified. Chemoprevention of lung cancer induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is discussed as an example of this approach.

Metabolism of food-derived heterocyclic amines in nonhuman primates

During the cooking of meats, several highly mutagenic heterocyclic amines (HCAs) are produced. Three HCAs, IQ, MeIQx, and PhIP have been under study for carcinogenicity in cynomolgus monkeys, and to date, IQ has been shown to be a potent hepatocarcinogen. Concomitantly, the metabolic processing of these HCAs has been examined. Metabolism studies show that the potent hepatocarcinogenicity of IQ is associated with the in vivo metabolic activation of IQ via N-hydroxylation and the formation of DNA adducts. In monkeys undergoing carcinogen bioassay with IQ, N-hydroxylation was confirmed by the presence of the N-hydroxy– N-glucuronide conjugate of IQ in urine. The N-hydroxylation of IQ appears to be carried out largely by hepatic CYP3A4 and/or CYP2C9/10, and not by CYP1A2, an isoform not expressed in liver of this species. Notably MeIQx is poorly activated in cynomolgus monkeys and lacks the potency of IQ to induce hepatocellular carcinoma after a 5-year dosing period. The poor activation of MeIQx appears to be due to the lack of constitutive expression of CYP1A2 and an inability of other cytochromes P450, such as CYP3A4 and CYP2C9/10, to N-hydroxylate the quinoxalines. MeIQx is detoxified in monkeys largely by conjugation with glucuronide at the N-1 position. Although the carcinogenicity of PhIP is not yet known, the metabolic data suggest that PhIP will be carcinogenic in this species. PhIP is metabolically activated in vivo in monkeys by N-hydroxylation, as discerned by the presence of the N-hydroxy– N-glucuronide conjugate in urine, bile, and plasma. PhIP also produces DNA adducts that are widely distributed in tissues. The results from these studies support the importance of N-hydroxylation in the carcinogenicity of HCAs in nonhuman primates and by analogy, the importance of this metabolic activation step in the possible carcinogenicity of dietary HCAs in humans.

Protective effects of diallyl sulfide on acetaminophen-induced toxicities

Diallyl sulfide (DAS), a major flavour component of garlic, is known to modulate drug metabolism and may protect animals from chemically induced toxicity and carcinogenesis. In this study the effects of DAS on the oxidative metabolism and hepatotoxicity induced by acetaminophen (APAP) in rats were investigated. In the hepatotoxicity evaluation of Fischer 344 rats there was a dose-dependent increase in the odds of mortality rate by APAP ( P = 0.009); DAS treatment significantly protected rats from APAP-related mortality ( P = 0.026). Liver toxicity determined by lactate dehydrogenase activity was significantly increased by APAP treatment (0.75 g/kg). Pretreatment with DAS protected animals from APAP-induced liver toxicity in a time- and dose-dependent fashion. Treatment of DAS (50 mg/kg) 3 hr after APAP dosing significantly ( P < 0.05) protected rats from APAP-induced liver toxicity. The metabolism of APAP (50 μM) in vitro was significantly inhibited by DAS (0.3−1 mM) in liver microsomes isolated from F344 rats. As the effect of DAS on APAP-induced hepatotoxicity in vivo was observed only when DAS was administered before or shortly after (<3 hr) APAP dosing, data suggested that the protective effect of DAS is mainly at the metabolic activation step of APAP. However, the possibility that DAS may also have effects on other drug metabolism systems, such as glutathione (GSH) and glutathione S-transferases, cannot be ruled out.

Cancer chemopreventive activity of brassinin, a phytoalexin from cabbage.

Brassinin [3-(S-methyldithiocarbamoyl)aminomethyl indole], a phytoalexin first identified as a constituent of cabbage, was synthesized and evaluated for cancer chemopreventive activity. Dose-dependent inhibition of 7,12-dimethylbenz[a]anthracene (DMBA)-induced preneoplastic lesion formation was observed with mouse mammary glands in organ culture, as was dose-dependent inhibition of DMBA-induced mouse skin tumors that were promoted by treatment with 12-O-tetradecanoylphorbol-13-acetate. Cyclobrassinin is a biologically derived product of the oxidative cyclization of brassinin, and was as active as the parent compound in inhibiting the formation of preneoplastic mammary lesions in culture; however, 2-methylbrassinin was not significantly active in this process. Therefore, oxidative cyclization may be an effective metabolic activation step. As judged by these tumor inhibition studies in conjunction with potential to induce phase II enzymes in mice or cell culture, brassinin may be effective as a chemopreventive agent during both the initiation and promotion phases of carcinogenesis. This is the first report documenting the chemopreventive potential of structurally novel indole-based phytoalexins that are naturally occurring in cruciferous vegetables, and the synthetic route described herein has proven amenable for scale-up production. The bifunctional structural nature of brassinin, bearing both an indole nucleus and a dithiocarbamoyl-aminomethyl moiety, is notably similar to the individual structural elements of other known chemopreventive agents such as indole-3-carbinol or benzylisothiocyanate. The favorable biological activity demonstrated by the compound may originate from the presence of these two moieties.

Mitochondrial formation of β-oxopropyl metabolites from bladder carcinogenic ω-carboxyalkylnitrosamines

Certain environmentally relevant nitrosamines specifically induce malignant tumors in the urinary bladder in several animal species. For this organotropic effect, formation of ω-carboxylated proximal metabolites has been found to be obligatory. The mechanism of action of these intermediates, however, is not yet clear. We investigated biotransformation of butyl-3-carboxypropylnitrosamine (CAS: 38252-74-3), methyl-3-carboxypropylnitrosamine (CAS: 61445-55-4) and methyl-5-carboxypentylnitrosamine by mitochondrial fractions from rat liver and renal cortex. On incubation with mitochondrial fractions, the respective β-oxidized metabolites butyl-2-oxopropylnitrosamine (CAS: 51938-15-9) or methyl-2-oxopropylnitrosamine (CAS: 55984-51-51) were formed. This biotransformation was ATP dependent, associated with the presence of mitochondrial marker enzyme (cytochrome c oxidase) in 7000 × g subfractions and was inhibited by octanoic acid. Highest metabolic rates were observed with rat liver fractions. These results demonstrate that ω-carboxylated nitrosamines are substrates for mitochondrial enzymes of fatty acid degradation, most probably following the degradation pathway of medium-chain fatty acids. By this reaction, water-soluble carboxylated nitrosamines of low genotoxic potential are converted into rather lipophilic 2-oxopropyl metabolites with high genotoxic and carcinogenic potency. In contrast to carboxylated metabolites, 2-oxopropyl derivatives are good substrates for cytochrome P-450 dependent monooxygenases. Therefore, mitochondrial β-oxidation appears to be an important step in metabolic activation of nitrosamines tumorigenic in the urinary bladder.

Inhibition of repairable DNA-damage in Escherichia coli K-12 cells recovered from various organs of nitrosamine-treated mice by vitamin A, phenethylisothiocyanate, oleic acid and triolein.

The influence of various dietary constituents--phenethylisothiocyanate (PEITC), oleic acid (OA), triolein (TO), and vitamin A (ROL)--on the genotoxic activity of nitrosamines (NDMA, NDELA, NPYR) was investigated. For this purpose differential DNA repair assays with Escherichia coli K-12 strains were performed in vitro and in vivo with mice. Under in vitro conditions (liquid holding), all compounds reduced nitrosamine induced DNA-damage in the indicator bacteria in the dose range 1-10 micrograms/ml, the ranking order of efficiency being PEITC greater than OA greater than ROL greater than or equal to TO. In animal-mediated assays, acute oral treatment with PEITC (17-150 mg/kg), 2 h before nitrosamine administration, resulted in a marked decrease of nitrosamine genotoxicity in liver, kidneys, lungs and in the blood. Also in other organs (spleen, testes) an increase in differential survival (which serves as a measure for repairable DNA damage) occurred. With ROL only a comparatively moderate antigenotoxic effect was obtained at a high dose level (250 mg/kg) under identical experimental conditions. OA (2000 mg/kg) and TO (16,000 mg/kg) were completely inactive. Upon repeated treatment (consecutive oral administration of the putative antigenotoxins over 4 days, a final treatment 24 h before nitrosamine administration) PEITC (150 mg/kg/day), ROL (80 mg/kg/day) and OA (2000 mg/kg/day) had no influence on the genotoxic effects of the nitrosamines. Repeated treatment with TO (4000-16,000 mg/kg/day) resulted in a moderate dose-dependent reduction of NDMA-induced DNA-damage in the indicator bacteria, whereas in combination with NPYR only a marginal effect was observed. Biochemical experiments indicated that the antigenotoxic effects of PEITC seen under in vivo conditions were due to inhibition of alpha-hydroxylation of the nitrosamines, whereas ROL and TO appeared not to interfere strongly with this metabolic activation step. Our results indicate that in vitro assays do only partly reflect the antigenotoxic properties of the different food constituents in vivo and that animal-mediated DNA repair assays with E. coli strains are an appropriate approach to study the effects of modifiers of nitrosamine genotoxicity in the living animal.

Metabolism of 4,4'-methylene-bis-2-chloroaniline (MOCA) by rats in vivo and formation of N-hydroxy MOCA by rat and human liver microsomes.

The metabolism of 4,4'-methylene-bis-2-chloroaniline (MOCA) was investigated because it is an animal carcinogen to which humans have been exposed. In CD rats, where MOCA is a hepatocarcinogen, less than or equal to 0.2% of an oral dose of [14C]MOCA was recovered unchanged in the urine; enzymatic hydrolysis and extraction of urinary radioactivity indicated the presence of glucuronide and sulfate conjugates. In rat bile, the predominant metabolite was N-glucuronyl MOCA. Liver microsomes from male CD rats or human males (surgical specimens) were incubated in vitro with [14C] MOCA. Metabolite formation, which was dependent upon reduced pyridine nucleotides and intact microsomes, was quantitated by TLC and HPLC using appropriate chemically synthesized standards. N-Hydroxylation of MOCA occurred at a rate of 335 +/- 119 pmol/min/mg rat microsomal protein (n = 3) versus 230 or 765 (n = 2) with microsomes from humans; the product was identified by isotopic dilution for both species. The rates of 5-hydroxy-MOCA (o-aminophenol) formation were 92 +/- 33 (rats) and 7, 35 (human); rates for the benzhydrol derivative were 82 +/- 12 (rats) and 60, 160 (human). In rats, all three rates were elevated 4- to 8-fold by pretreatment with phenobarbital, which also enhanced the formation of partially characterized polar derivatives that appeared to result from oxidation and cleavage at the methylene carbon. The latter pathway typically amounted to 50-100% of the 4,4'-diamino-3,3'-dichlorobenzhydrol value in control or pretreated animals. Thus, rats metabolize MOCA extensively and the pathways include N-hydroxlation, which is regarded as an obligatory step in metabolic activation of arylamines. The presence of MOCA N-hydroxylase in human liver supports the hypothesis that exposure of humans to MOCA entails a carcinogenic risk.

The S-acetyl coenzyme A-dependent metabolic activation of the carcinogen N-hydroxy-2-aminofluorene by human liver cytosol and its relationship to the aromatic amine N-acetyltransferase phenotype.

A genetic polymorphism in the enzymatic N-acetylation of sulfamethazine and other drugs in humans is well known and has been related to differential susceptibility to drug toxicities. Carcinogenic aromatic amines such as 2-aminofluorene also undergo N-acetylation, and phenotypic slow acetylator individuals have been suggested to be at increased risk to arylamine-induced urinary bladder cancer. However, acetyltransferases have also been shown to catalyze a final metabolic activation step in the conversion of both hydroxamic acid (e.g. N-hydroxy-N-acetyl-2-aminofluorene N,O-acyltransferase) and N-hydroxy-arylamine (e.g. N-hydroxy-2-aminofluorene O-acetyltransferase) metabolites to DNA-bound adducts. In this regard, rapid acetylators have recently been reported to be at higher risk for colorectal cancer. In this study, we examined the enzymatic activity of 35 human liver cytosol samples (obtained surgically from organ donors) for sulfamethazine and 2-aminofluorene N-acetyltransferase activities, N-hydroxy-N-acetyl-2-aminofluorene N,O-acyltransferase activity, and the acetyl coenzyme A (CoA)-dependent O-acetylation of N-hydroxy-2-aminofluorene to form DNA-bound products. The results with sulfamethazine indicated that about two-thirds of the human liver samples were of the slow acetylator phenotype; the same individuals also exhibited levels of 2-aminofluorene N-acetylation that were consistent with their respective sulfamethazine-N-acetylation activity. N-Hydroxy-N-acetyl-2-aminofluorene N,O-acyltransferase activity was not detected. However, the acetyl CoA-dependent activation of N-hydroxy-2-aminofluorene was observed for nearly all of the samples and was consistently higher in the fast acetylator group. These data support the hypothesis that phenotypic rapid acetylator individuals are likely to be at higher risk to aromatic amine-induced cancers in those tissues containing appreciable levels of N-hydroxy arylamine O-acetyltransferase.

Metabolic aspects of pyrolysis mutagens in food.

The first step in metabolic activation of mutagenic and carcinogenic heterocyclic amines has been elucidated to be N-hydroxylation by cytochrome P-448. N-Hydroxyamino compounds are further activated to form N-O-acyl derivatives that readily react with DNA. The adducts between the metabolites of Trp-P-2 and Glu-P-1 and DNA were shown to have a C8-guanylamino structure. In the case of Glu-P-1, modification of guanine in GC clusters occurred preferentially. Glutathione transferases and myeloperoxidase were shown to inactivate some heterocyclic amines or their active metabolites. Hemin and fatty acids bind to and inactivate them. Fibers and other factors from vegetables also work to inactivate heterocyclic amines. Nitrite at low pH also degraded some heterocyclic amines, but those with an imidazole moiety were resistant. Glu-P-1 induced intestinal tumors in a high incidence when fed orally to rats. When 14C-Glu-P-1 was administered by gavage into rats about 50% and 35% were excreted into feces and urine, respectively, within 24 hr. When the bile was collected, around 60% of radioactivity was excreted into it within 24 hr. In the bile, N-acetyl-Glu-P-1 was identified as one of the metabolites of Glu-P-1. It showed a mutagenic activity of about one fourth that of Glu-P-1 with S9 mix. Some radioactivity was also detected in the blood. At 24 hr after administration, most of the radioactivity was found to be bound to erythrocyte beta-globins and serum proteins including albumin.

Genetic effects of isoniazid and the relationship to in vivo and in vitro biotransformation

The mutagenic activity of isoniazid, N-acetyl-isoniazid and hydrazine dihydrochloride was investigated in S. typhimurium. Isoniazid was found to possess a weak mutagenic activity only in repair-deficient strains TA1535 and TA100 as well as in the plasmid-containing strain TA92 (10–30 mg/plate) in the Ames test without metabolic activation. Addition of microsomal enzymes by S9 mix decreased this direct mutagenic activity. In contrast, preincubation of isoniazid with crude liver homogenate from mice, rats or Syrian golden hamsters for 4 h prior to plating with bacteria liberated a mutagenic compound which is equally active in both repair-deficient and repair wild-type strains (0.5–5 mg/plate). This activation pathway is independent of NADPH, is heat-sensitive and is operative only in a total liver homogenate in suspension. The highest capacity for mutagenic activation was achieved with liver homogenate from hamsters, followed by that from mice and rats. Furthermore, this mutagenic activation is paralleled by formation of hydrazine, as demonstrated in colorimetric measurements with p-dimethylaminobenzaldehyde. N-Acetyl-isoniazid is without mutagenic activity under similar conditions, and liberation of hydrazine was never detected. This means that, besides having a weak direct genetic activity, isoniazid is a promutagen, and formation of hydrazine is the first step in metabolic activation. It is concluded that the genotoxic properties of isoniazid in mammals are primarily determined by the pharmacokinetic behavior of the ultimate reactive metabolite. This result must be taken into consideration in risk assessment performed for mutagenic and carcinogenic properties of isoniazid in man.

Studies on chemical carcinogens. XXIV. Mutagenic potency of alkylated 4-nitroquinoline 1-oxides: its dependence on the rate of metabolic activation.

Twelve kinds of alkylated derivatives of 4-nitroquinoline 1-oxide (4NQO) were tested for their mutagenicity on S. typhimurium TA100. The assay method was slightly modified from the original procedure of Ames in order to obtain a better correlation with intrinsic chemical properties of the compounds. It was found that mutagenic potencies of these derivatives were linearly correlated with the metabolic rates of 4NQO's to the corresponding 4-hydroxyaminoquinoline 1-oxides (4HAQO's), the correlation coefficient being 0.930. The steric requirements for enzymic reduction of the nitro group seem to be the major determinant of the mutagenic activity of the derivatives, and surprisingly, the substituents do not have any appreciable effect either on the subsequent metabolic activation step (aminoacylation of 4HAQO's) or on the ultimate nonenzymatic modification step.

New Approaches to Immunotoxicity Testing

New approaches to immunotoxicity testing are reviewed and discussed. A method of activating T-cells in vivo is presented which circumvents artifacts dur to viability effects encountered with in vitro mitogen assays. The use of adoptive transfer approaches to combine the advantages of in vitro manipulation with in vivo function assays is discussed relative to natural killer cells. The need for an in vitro metabolic activation step coupled to other in vitro immunologic assays is discussed.

New approaches to immunotoxicity testing.

New approaches to immunotoxicity testing are reviewed and discussed. A method of activating T-cells in vivo is presented which circumvents artifacts dur to viability effects encountered with in vitro mitogen assays. The use of adoptive transfer approaches to combine the advantages of in vitro manipulation with in vivo function assays is discussed relative to natural killer cells. The need for an in vitro metabolic activation step coupled to other in vitro immunologic assays is discussed.