S9 Preparations Research Articles

Addition of Phenylmethylsulfonyl Fluoride Increases the Working Lifetime of the Trout Liver S9 Substrate Depletion Assay, Resulting in Improved Detection of Low Intrinsic Clearance Rates.

The activity of a trout liver S9 substrate depletion assay has been shown to decline over time, presumably due to proteolytic degradation of biotransformation enzymes. To address this problem, assay performance was evaluated following the addition of phenylmethylsulfonyl fluoride (PMSF) or a general-purpose protease inhibitor cocktail to liver homogenization buffers and/or S9 reaction mixtures. Addition of PMSF to liver homogenization buffers and/or S9 reaction mixtures had little or no effect on clearance of phenanthrene, a model cytochrome P450 substrate, in short-term (25 or 30 min) depletion experiments but resulted in significant improvements in retention of this initial activity over time. The protease inhibitor cocktail strongly inhibited initial activity when added to homogenization buffers or reaction mixtures. Taking into consideration potential effects on liver carboxylesterases, the treatment approach determined to be optimal was addition of 10 µM PMSF to the S9 reaction mixture. Addition of 10 µM PMSF to the mixture resulted in significantly higher rates of phenanthrene clearance in 2-h incubations relative to those obtained in the absence of PMSF and a 6-fold increase in the working lifetime of the preparation. The results of a statistical power analysis suggest that by increasing the working lifetime of the assay, addition of PMSF to the reaction mixture could result in substantially improved detection of low in vitro clearance rates when compared to current practice. These findings demonstrate the value of adding PMSF to the trout S9 preparation and may have broad implications for use of this assay to support chemical bioaccumulation assessments for fish. Environ Toxicol Chem 2021;40:148-161. © 2020 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Reductive Metabolism Influences the Toxicity and Pharmacokinetics of the Hypoxia-Targeted Benzotriazine Di-Oxide Anticancer Agent SN30000 in Mice.

3-(3-Morpholinopropyl)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine 1,4-dioxide (SN30- 000), an analog of the well-studied bioreductive prodrug tirapazamine (TPZ), has improved activity against hypoxic cells in tumor xenografts. However, little is known about its biotransformation in normal tissues. Here, we evaluate implications of biotransformation of SN30000 for its toxicokinetics in NIH-III mice. The metabolite profile demonstrated reduction to the 1-N-oxide (M14), oxidation of the morpholine side-chain (predominantly to the alkanoic acid M18) and chromophore, and subsequent glucuronidation. Plasma pharmacokinetics of SN30000 and its reduced metabolites was unaffected by the presence of HT29 tumor xenografts, indicating extensive reduction in normal tissues. This bioreductive metabolism, as modeled by hepatic S9 preparations, was strongly inhibited by oxygen indicating that it proceeds via the one-electron (radical) intermediate previously implicated in induction of DNA double strand breaks and cytotoxicity by SN30000. Plasma pharmacokinetics of SN30000 and M14 (but not M18) corresponded closely to the timing of reversible acute clinical signs (reduced mobility) and marked hypothermia (rectal temperature drop of ∼8°C at nadir following the maximum tolerated dose). Similar acute toxicity was elicited by dosing with TPZ or M14, although M14 did not induce the kidney and lung histopathology caused by SN30000. M14 also lacked antiproliferative potency in hypoxic cell cultures. In addition M14 showed much slower redox cycling than SN30000 in oxic cultures. Thus a non-bioreductive mechanism, mediated through M14, appears to be responsible for the acute toxicity of SN30000 while late toxicities are consistent with DNA damage resulting from its one-electron reduction. A two-compartment pharmacokinetic model, in which clearance of SN30000 is determined by temperature-dependent bioreductive metabolism to M14, was shown to describe the non-linear PK of SN30000 in mice. This study demonstrates the importance of non-tumor bioreductive metabolism in the toxicology and pharmacokinetics of benzotriazine di-oxides designed to target tumor hypoxia.

Measurement of kinetic parameters for biotransformation of polycyclic aromatic hydrocarbons by trout liver S9 fractions: Implications for bioaccumulation assessment.

In vitro substrate depletion methods developed by the pharmaceutical industry are being used with increasing frequency to support chemical bioaccumulation assessments for fish. However, the application of these methods to high log K ow chemicals poses special challenges. Biotransformation of three polycyclic aromatic hydrocarbons (PAHs) was measured using trout liver S9 fractions. Measured activity declined with incubation time and was reduced by acetone (used as a spiking solvent) at concentrations greater than 0.5%. Addition of alamethicin, a pore-forming peptide used to support UDP-glucuronosyltransferase activity, also reduced activity in a concentration-dependent manner. The substrate concentration dependence of activity was evaluated to estimate K M and V max values for each compound. Derived kinetic constants suggested that all three PAHs are transformed by the same reaction pathway and indicated an inverse correlation between K M and chemical log K ow. Binding effects on activity were evaluated by measuring unbound chemical concentrations across a range of S9 protein levels. Reaction rates were proportional to the unbound concentration except when these concentrations approached saturating levels, providing a direct demonstration of the free chemical hypothesis. These findings suggest that previous in vitro work with high log K ow compounds was conducted at inappropriately high substrate concentrations resulting in underestimation of true in vivo activity. Preliminary calculations also indicate that PAH metabolism in fish may approach saturation during standardized in vivo testing efforts, potentially resulting in concentration-dependent accumulation and/or steady-state levels of accumulation greater than those which occur in a natural setting.

The utility of metabolic activation mixtures containing human hepatic post-mitochondrial supernatant (S9) for in vitro genetic toxicity assessment.

In vitro genotoxicity assessment routinely employs an exogenous metabolic activation mixture to simulate mammalian metabolism. Activation mixtures commonly contain post-mitochondrial liver supernatant (i.e. S9) from chemically induced Sprague Dawley rats. Although Organization for Economic Cooperation and Development (OECD) test guidelines permit the use of other S9 preparations, assessments rarely employ human-derived S9. The objective of this study is to review and evaluate the use of human-derived S9 for in vitro genetic toxicity assessment. All available published genotoxicity assessments employing human S9 were compiled for analysis. To facilitate comparative analyses, additional matched Ames data using induced rat liver S9 were obtained for certain highly cited chemicals. Historical human and induced rat S9 quality control reports from Moltox were obtained and mined for enzyme activity and mutagenic potency data. Additional in vitro micronucleus data were experimentally generated using human and induced rat S9. The metabolic activity of induced rat S9 was found to be higher than human S9, and linked to high mutagenic potency results. This study revealed that human S9 often yields significantly lower Salmonella mutagenic potency values, especially for polycyclic aromatic hydrocarbons, aflatoxin B1 and heterocyclic amines (~3- to 350-fold). Conversely, assessment with human S9 activation yields higher potency for aromatic amines (~2- to 50-fold). Outliers with extremely high mutagenic potency results were observed in the human S9 data. Similar trends were observed in experimentally generated mammalian micronucleus cell assays, however human S9 elicited potent cytotoxicity L5178Y, CHO and TK6 cell lines. Due to the potential for reduced sensitivity and the absence of a link between enzyme activity levels and mutagenic potency, human liver S9 is not recommended for use alone in in vitro genotoxicity screening assays; however, human S9 may be extremely useful in follow-up tests, especially in the case of chemicals with species-specific metabolic differences, such as aromatic amines.

Effect of structural modification on the gastrointestinal stability and hepatic metabolism of α-aminoxy peptides

α-Aminoxy peptide AxyP1 has been reported to form synthetic chloride channel in living cells, thus it may have therapeutic potential for the treatment of diseases associated with chloride channel dysfunction. However, this study revealed significant gastrointestinal (GI) instability and extensive hepatic metabolism of AxyP1. To improve its GI and metabolic stability, structural modifications were conducted by replacing the isobutyl side chains of AxyP1 with methyl group (AxyP2), hydroxymethyl group (AxyP3), 4-aminobutyl group (AxyP4) and 3-carboxyl propyl group (AxyP5). Compared with AxyP1 (41 and 47 % degradation), GI stability of the modified peptides was significantly improved by 8-fold (AxyP2), 9-fold (AxyP3) and 12-fold (AxyP5) with no degradation for AxyP4 in simulated gastric fluid within 1 h, and by 12-fold (AxyP2) and 9-fold (AxyP3) with no degradation for AxyP4 and AxyP5 in simulated intestinal fluid within 3 h, respectively. The hepatic metabolic stability of the four modified peptides within 30 min in rat liver S9 preparation was also improved significantly with no metabolism of AxyP5 and threefold (AxyP2 and AxyP4) and eightfold (AxyP3) less metabolism compared with AxyP1 (39 % metabolism). Unlike hydrolysis as the major metabolism of peptides of natural α-amino acids, oxidation mediated by the cytochrome P450 enzymes, especially CYP3A subfamily, to form the corresponding mono-hydroxyl metabolites was the predominant hepatic metabolism of the five α-aminoxy peptides tested. The present findings demonstrate that structural modification can significantly improve the GI and metabolic stability of α-aminoxy peptides and thus increase their potential for therapeutic use in the treatment of chloride channel related diseases.

Preclinical pharmacology of novel indolecarboxamide ML-970, an investigative anticancer agent.

ML-970 (AS-I-145; NSC 716970) is an indolecarboxamide synthesized as a less toxic analog of CC-1065 and duocarmycin, a natural product that binds the A-T-rich DNA minor groove and alkylates DNA. The NCI60 screening showed that ML-970 had potent cytotoxic activity, with an average GI(50) of 34 nM. The aim of this study is to define the pharmacological properties of this novel anticancer agent. We established an HPLC method for the compound, examined its stability, protein binding, and metabolism by S9 enzymes, and conducted pharmacokinetic studies of the compound in two strains of mice using two different formulations. ML-970 was relatively stable in plasma, being largely intact after an 8-h incubation in mouse plasma at 37°C. The compound was extensively bound to plasma proteins. ML-970 was only minimally metabolized by the enzymes present in S9 preparation and was not appreciably excreted in the urine or feces. The solution formulation provided higher C(max), AUC, F values, and greater bioavailability, although the suspension formulation resulted in a later T(max) and a slightly longer T(1/2). To determine the fate of the compound, we accomplished in-depth studies of tissue distribution; the results indicated that the compound undergoes extensive enterohepatic circulation. The results obtained from this study will be relevant to the further development of the compound and may explain the lower myelotoxicity of this analog compared to CC-1065.

Overcoming the Genotoxicity of a Pyrrolidine Substituted Arylindenopyrimidine As a Potent Dual Adenosine A2A/A1 Antagonist by Minimizing Bioactivation to an Iminium Ion Reactive Intermediate

2-Amino-4-phenyl-8-pyrrolidin-1-ylmethyl-indeno[1,2-d]pyrimidin-5-one (1) is a novel and potent selective dual A(2A)/A(1) adenosine receptor antagonist from the arylindenopyrimidine series that was determined to be genotoxic in both the Ames and Mouse Lymphoma L5178Y assays only following metabolic activation. Compound 1 was identified as a frame-shift mutagen in Salmonella typhimurium tester strain TA1537 as indicated by a significant dose-dependent increase in revertant colonies as compared to the vehicle control. The metabolic activation-dependent irreversible covalent binding of radioactivity to DNA, recovery of 1 and its enamine metabolite from acid hydrolysis of covalently modified DNA, and protection of covalent binding to DNA by both cyanide ion and methoxylamine suggest that the frame-shift mutation in TA1537 strain involved covalent binding instead of simple intercalation to DNA. Compound 1 was bioactivated to endocyclic iminium ion, aldehyde, epoxide, and α,β-unsaturated keto reactive intermediates from the detection of cyano, oxime, and glutathione conjugates by data-dependent high resolution accurate mass measurements. Collision-induced dissociation of these conjugates provided evidence for bioactivation of the pyrrolidine ring of 1. The epoxide and α,β-unsaturated keto reactive intermediates were unlikely to cause the genotoxicity of 1 because the formation of their glutathione adducts did not ameliorate the binding of compound related material to DNA. Instead, the endocyclic iminium ions and amino aldehydes were likely candidates responsible for genotoxicity based on, first, the protection afforded by both cyanide ion and methoxylamine, which reduced the potential to form covalent adducts with DNA, and, second, analogues of 1 designed with low probability to form these reactive intermediates were not genotoxic. It was concluded that 1 also had the potential to be mutagenic in humans based on observing the endocyclic iminium ion following incubation with a human liver S9 preparation and the commensurate detection of DNA adducts. An understanding of this genotoxicity mechanism supported an evidence-based approach to selectively modify the structure of 1 which resulted in analogues being synthesized that were devoid of a genotoxic liability. In addition, potency and selectivity against both adenosine A(2A) and A(1) receptors were maintained.

Assessment of thyroid hormone activity of halogenated bisphenol A using a yeast two-hybrid assay

The thyroid hormone agonist/antagonist activities of halogenated derivatives of bisphenol A (BPA) were assessed using a yeast two-hybrid assay incorporating the human thyroid hormone α (TR α), both with and without possible metabolic activation by rat liver S9 preparation. In the absence of the rat liver S9 preparation, 3,3′,5,5′-tetrabromobisphenol A (TBBPA), 3,3′,5,5′-tetrachlorobisphenol A (TCBPA), and 3,3′,5-trichlorobisphenol A (3,3′,5-triClBPA) exhibited agonist activity, whereas 3-chlorobisphenol A (3-ClBPA), 3,5-dichlorobisphenol A (3,5-diClBPA), 3,3′-dichlorobisphenol A (3,3′-diClBPA), and BPA did not. The activities of TBBPA and TCBPA increased markedly (7.6-fold and 3.1-fold, respectively) after their metabolic activation with the rat liver S9 preparation. TBBPA, TCBPA, and 3,3′,5-triClBPA inhibited the binding of triiodothyronine (T3) to TR α at 2 × 10 −5 M without rat liver S9 treatment and 4 × 10 −6 M with rat liver S9 treatment, demonstrating their T3 antagonist activity. These results revealed that metabolic activation by rat liver S9 significantly increased the agonist/antagonist potential of some halogenated BPAs.

Recombinant human cytochrome P450 monooxygenases for drug metabolite synthesis

Cytochrome P450 monooxygenases (CYPs) are important enzymes in the metabolism of xenobiotics. Therefore, several approaches to clone and overexpress the human isoforms have been made. In addition to microsomes or S9 preparations, these recombinant human isoforms have found diverse application in drug development. We discuss and give examples of the use of bacterial whole cell systems with rec. human CYPs for the preparative scale synthesis of drug metabolites.

Nonclinical pharmacokinetics of BMS‐292655, a water‐soluble prodrug of the antifungal ravuconazole

The phosphate ester, BMS-292655, was developed as a water-soluble prodrug of the antifungal agent, ravuconazole (BMS-207147). BMS-292655 was comparatively stable in rat, beagle dog, cynomolgus monkey and human plasma, but was hydrolysed upon incubation with liver S9 preparations from all species. The major product in rat, monkey and human S9 was BMS-207147, while in dog S9, the intermediate ester, BMS-300043, predominated. BMS-300043 itself was more stable in dog S9 than in S9 preparations from the other species. Intravenous administration of BMS-292655 to rats, beagle dogs and cynomolgus monkeys indicated species differences in the extent of formation of BMS-207147 (monkeys>rats>dogs). The lower overall generation of BMS-207147 in dogs was consistent with the presence of circulating plasma levels of BMS-300043. BMS-300043 was present in monkey plasma but not detectable in rat plasma. The conversion of BMS-292655 to BMS-207147 in the presence of human S9 indicated the potential for BMS-292655 to function as a BMS-207147 prodrug in humans. The similarity in the hydrolysis of BMS-292655 when incubated with human and monkey S9 in vitro, coupled with the effective release of BMS-207147 from BMS-292655 upon i.v. administration to monkeys, is consistent with this conclusion.

Effect of Resveratrol on 17β-Estradiol Sulfation by Human Hepatic and Jejunal S9 and Recombinant Sulfotransferase 1E1

The purpose of this study was to investigate the sulfation of resveratrol (3,5,4'-trihydroxystilbene) and its potential to exhibit drug-drug interactions via sulfation. The possible interaction of resveratrol with 17beta-estradiol (E2), a major estrogen hormone and prototypic substrate for sulfate conjugation, was studied. Resveratrol and E2 are both known to undergo sulfate conjugation catalyzed by human sulfotransferases (SULTs). Resveratrol is a phytoestrogen with mixed estrogen agonist/antagonist properties that is being developed as a chemopreventive agent. The sulfate conjugation of E2 and resveratrol were studied individually using S9 fractions from human liver and jejunum as well as recombinant human SULT isoforms. The sulfation of E2 (3-20 nM) was then investigated in the presence of various concentrations (0, 0.5, 1, and 2 microM) of resveratrol using the two S9 preparations as well as recombinant SULT1E1, the major isoform responsible for E2 sulfation. Resveratrol inhibited E2 sulfation with estimated K(i) values of 1.1 microM (liver), 0.6 microM (jejunum), and 2.3 microM (SULT1E1), concentrations that could be pharmacologically relevant. The results suggest that these phytoestrogens can potentially alter the homeostasis of estrogen levels. These findings also imply that resveratrol may inhibit the metabolism of other estrogen analogs or therapeutic agents such as ethinylestradiol or dietary components that are also substrates for SULT1E1.

Hepatic nitroreduction, toxicity and toxicokinetics of the anti-tumour prodrug CB 1954 in mouse and rat

5-(Aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954), a promising anti-tumour compound, is associated with clinical hepatotoxicity. We have previously demonstrated that human liver preparations are capable of endogenous 2- and 4-nitroreduction of CB 1954 to generate highly potent cytotoxins. The present study initially examined the in vitro metabolism of CB 1954 in S9 preparations of several non-clinical species and strains. The CD-1 nu/ nu mouse and Sprague–Dawley rat were subsequently chosen for further assessment of in vivo metabolism and hepatotoxicity of CB 1954, as well as the mechanisms that may be involved. Animals were administered the maximum tolerated dose (MTD). At 562 μmol/kg, the mouse exhibited transaminase elevation and centrilobular hepatocyte injury. Moreover, thiol adducts as well as hepatic glutathione depletion paralleled temporally by maximal nitroreduction were observed. The rat had a much lower MTD of 40 μmol/kg and showed signs of gastro-intestinal disturbances. In contrast to mouse, peri-portal damage and biliary changes were observed in rat without any alterations in plasma biomarkers or hepatic glutathione levels. Immunohistochemical analysis did not reveal any correlation between the location of injury and expression of cytochrome P450 reductase and NAD(P)H quinone oxidoreductase 1, two enzymes implicated in the bioactivation of this drug. In conclusion, the present study showed that following administration of CB 1954 at the respective MTDs, hepatotoxicity was observed in both mouse and rat. However, the degree of sensitivity to the drug and the mechanisms of toxicity involved appear to be widely different between CD-1 nu/ nu mice and Sprague–Dawley rats.

1,2,3-Thiadiazole substituted pyrazolones as potent KDR/VEGFR-2 kinase inhibitors

KDR kinase inhibition is considered to play an important role in regulating angiogenesis, which is vital for the survival and proliferation of tumor cells. Recently we disclosed a structure-based kinase inhibitor design strategy which led to the identification of a new class of VEGFR-2/KDR kinase inhibitors bearing heterocyclic substituted pyrazolones as the core template. Instability in a rat S9 preparation and poor iv PK profiles for most of these inhibitors necessitated exploration of new pyrazolones to identify new analogs with improved metabolic stability. Optimization of the heterocyclic moiety led to the identification of the thiadiazole series of pyrazolones ( D) as potent VEGFR-2/KDR kinase inhibitors. SAR modifications, kinase selectivity profiling, and structural elements for improved PK properties were explored. Oral bioavailability up to 29% was achieved in the rat. Modeling results based on the Glide XP docking approach supported our postulation regarding the interaction of the lactam segment of the pyrazolones with the hinge region of the KDR kinase.

Modulation of hepatic cytochromes P450 and phase II enzymes by dietary doses of sulforaphane in rats: Implications for its chemopreventive activity

The principal objectives of our study were to ascertain whether sulforaphane, at dietary levels of intake, modulates rat hepatic cytochrome P450 and phase II enzyme systems and to evaluate the impact of such changes in the chemopreventive activity of this isothiocyanate. Animals were exposed to sulforaphane in their drinking water for 10 days, equivalent to daily doses of 3 and 12 mg/kg. Depentylation of pentoxyresorufin decreased and was paralleled by a decline in CYP2B apoprotein levels. At the higher dose, erythromycin N-demethylase activity declined and was accompanied by a similar decrease in CYP3A2 apoprotein levels. However, sulforaphane treatment upregulated CYP1A2 levels, determined immunologically, but the dealkylations of methoxy- and ethoxyresorufin were not similarly increased. Hepatic S9 preparations from sulforaphane-treated rats were less effective than control preparations in converting IQ (2-amino-3-methylimidazo-[4,5-f]quinoline) to mutagenic intermediates in the Ames test. To clarify the underlying mechanism, in vitro studies were undertaken. In beta-naphthoflavone-treated rats, the inhibition by sulforaphane of the O-dealkylations of methoxy- and ethoxyresorufin was enhanced if the isothiocyanate was preincubated in the presence of NADPH. It may be inferred that sulforaphane induces hepatic CYP1A2 but the enzyme is not catalytically competent because of bound sulforaphane metabolite(s). Finally, sulforaphane stimulated, in a dose-dependent fashion, quinone reductase but failed to influence glutathione S-transferase, epoxide hydrolase and glucuronosyl transferase activities. It is concluded that, even at dietary doses, sulforaphane can modulate the xenobiotic-metabolising enzyme systems, shifting the balance of carcinogen metabolism toward deactivation, and this may be an important mechanism of its chemopreventive activity.

Estrogenic and thyroid hormone activity of a series of hydroxy-polychlorinated biphenyls

A series of novel synthetic monohydroxy polychlorinated biphenyls (OH-PCBs) (5 trichloro-, 5 tetrachloro- and 5 pentachloro-compounds) have been characterized (1H and 13C NMR and high resolution MS) and their estrogenic and thyroid hormone activities assessed using a yeast two-hybrid assay, both with and without possible metabolic activation by rat liver S9 preparation. Moderate estrogenic activity was found for 2,3,4′-trichlorobiphenyl-4-ol (compound 5) but this was eliminated when exposed to the S9 mix. 2,2′,3′,4,6-Pentachlorobiphenyl-3-ol (13) and 2′,3,3′,6-tetrachlorobiphenyl-4-ol (10) both showed weak estrogenicity in the absence of the S9 mix. The estrogenicity of compound (10) was enhanced 10-fold by exposure to S9 metabolic activation but that of compound (13) remained unchanged. 2′,4,5′,6-Tetrachlorobiphenyl-2-ol (6) showed strong thyroid hormonal activity (5% of that of T4) whereas 3′,4,6-trichlorobiphenyl-3-ol (4), compound (10) and 2,3′,4,5′,6-pentachlorobiphenyl-3-ol (14) showed moderate activity, and 2′,3,3′,5-tetrachlorobiphenyl-2-ol (8) and 3,3′,5,5′,6-pentachlorobiphenyl-2-ol (11) showed weak activity. The activity of (4) was eliminated by S9 metabolic activation whereas those of (6) and (14) were weakened and that of (10) remained unchanged.

An improvement of the Ames test using a modified human liver S9 preparation

Introduction: In preliminary studies on the Ames test using human liver S9 fractions, we found that the crude human S9 fractions, obtained following centrifugation of the tissue homogenate for 20 min at 9000× g, were not always sterile. When this was the case, the S9 fractions were often accompanied by an increased number of colonies above the normal range on plates in the solvent control used in the Ames test. In addition, we also sometimes identified the incorporation of a small amount of fat in the crude human liver S9 fractions. We have therefore obtained a purified fat-free S9 fraction by a simple modification to the crude S9 preparation; fat was completely removed by centrifugation of the crude S9 fraction. Methods: Using the purified and crude human S9 fractions (two lots each), both the sterility and the number of bacterial colonies produced on a plate with five bacterial tester strains by solvent controls (purified water and dimethyl sulfoxide) were examined. The findings were then compared to those observed with phosphate buffer or S9 fraction from rats pretreated with phenobarbital/5,6-benzoflavone. Results: The data show that each of the crude human S9 fractions was not sterile and produced an increasing number of colonies with each solvent control, almost equal to the sum of the numbers of contaminating bacterial colonies and spontaneous revertant colonies observed with phosphate buffer or the rat S9 fraction. On the other hand, both the purified human S9 fractions were sterile, and the number of colonies that appeared in each solvent control was similar to that of spontaneous revertant colonies observed with phosphate buffer or the rat S9 fraction. Discussion: These results indicate that this new procedure of S9 preparation, modified with an additional recentrifugation step, may provide a high quality of purified fat- and bacteria-free S9 fraction for use in the Ames test.

Using base-specific Salmonella tester strains to characterize the types of mutation induced by benzidine and benzidine congeners after reductive metabolism

Although benzidine (Bz), 4-aminobiphenyl (ABP), 3,3′-dichlorobenzidine HCl (DCBz), 3,3′-dimethylbenzidine (DMBz), 3,3′-dimethoxybenzidine (DMOBz) and the benzidine congener-based dye trypan blue (TB) produce primarily frameshift mutations in Salmonella typhimurium, the base-substitution strain TA100 also responds to these compounds when S9 is present. Performing DNA sequence analysis, other investigators have shown that ABP induces frameshift, base-pair and complex mutations. Also, it was found that an uninduced hamster liver S9 preparation with glucose-6-phosphate dehydrogenase, FMN, NADH and four times glucose 6-phosphate gave a stronger mutagenic response than the conventional plate incorporation with rat S9 activation mixture for all the compounds tested. Using the base-specific tester strains of S. typhimurium (TA7001–TA7006) with the above reductive metabolic activation system, we surveyed these compounds for the ability to produce specific base-pair substitutions after reductive metabolism. Bz was weakly mutagenic in TA7005 (0.04 revertants/μg). ABP was mutagenic in TA7002 (1.4 revertants/μg), TA7004 (0.6 revertants/μg), TA7005 (2.98 revertants/μg) and TA7006 (0.4 revertants/μg). DCBz was weakly mutagenic in TA7004 (0.01 revertants/μg). It was concluded that benzidine induced some CG->AT transversions in addition to frameshift mutations. ABP induced TA->AT, CG->AT, and CG->GC transversions as well as GC->AT transitions. DCBz induced only GC->AT transitions. Because DMBz, DMOBz and TB were not mutagenic in this base-substitution mutagen detection system, their mutagenic activity was attributed strictly to frameshift mechanisms.

Cytotoxicity of settling particulate matter and sediments of the Neckar River (Germany) during a winter flood

AbstractTo investigate the cytotoxic and genotoxic potentials of settling particulate matter (SPM) carried by the Neckar River, a well‐studied model for a lock‐regulated river in central Europe, during a flood, acute cytotoxicity was investigated using the fibroblast‐like fish cell line RTG‐2 with the neutral red retention, the succinic acid dehydrogenase (MTT), and the lactatedehydro‐genase (LDH) release assays as well as microscopic inspection as endpoints. Genotoxicity of water, pore water, sediments, and SPM were assessed using the Ames test. Different extraction methods (Soxhlet extraction with solvents of variable polarity as well as a fluid/fluid extraction according to pH) in addition to a supplementation of biotests with S9 fractions from the liver of β‐naphthoflavone/phenobarbital‐induced rats allowed a further characterization of the biological damage. Both sediments and SPM extracts caused cytotoxic effects in RTG‐2 cells. Cytotoxicity was found to increase significantly with polarity of extracting solvents (NR50 = effective concentration for 50% cell death in the neutral red test: 80 [65], 100 [70], 180 [220], and 225 [270] mg/ml for ethanol, acetone, dichloromethane, and n‐hexane extracts, respectively, if measured with [without] S9 supplementation). Following extraction according to pH, cytotoxicity could be attributed mainly to neutral substances (NR50: 80 and 218 mg dry SPM/ml test medium for the neutral and the acid fractions, respectively), whereas the slightly acid and basic fractions already showed little or no cytotoxicity. Samples taken during the period of flood rise showed the highest cytotoxic activities. Cytotoxicity was significantly enhanced by the addition of S9 preparations. In contrast, no genotoxic activity was found in native surface waters, pore waters, and SPM.

IN VITRO INHIBITION OF CARCINOGEN-INDUCED MUTAGENICITY BY CASSIA OCCIDENTALIS AND EMBLICA OFFICINALIS

Aqueous extracts of Cassia occidentalis Linn. (Leguminoceae) and Emblica officinalis Gaertn. (Euphorbiaceae) were screened for effectiveness in inhibiting mutagenicity of aflatoxin B1 (AFB1) and benzo[a]-pyrene (B[a]P) in the Ames test. Antimutagenicity was evaluated using Salmonella typhimurium strains TA 98 and TA 100. In the assay, metabolic activation of AFB1 (0.5 μg/plate) and B[a]P (1 μg/plate) was mediated by rat liver S9 preparation. Although both plants inhibited mutagenicity, E. officinalis had more inhibitory effect than C. occidentalis. Their action is possibly mediated through interactions with micro somal activating enzymes. Their inhibitory action on chromosomal aberrations together with present results suggest that these plants have potent antimutagenic and anticarcinogenic activities against mutagens requiring metabolic activation.

Identification of in vitro metabolites of indinavir by “intelligent automated LC-MS/MS” (INTAMS) utilizing triple quadrupole tandem mass spectrometry

In an effort to improve the efficiency of the TSQ 7000 LC-MS/MS system for identification of drug metabolites in biological matrices in support of drug discovery programs, a combination of instrument control language procedures for the Finnigan MAT TSQ 7000 mass spectrometer, referred to as INTAMS, were composed. INTAMS was designed to conduct unattended, automatic liquid chromatography/mass spectrometry (LC-MS) and LC-MS/MS analyses of drugs and metabolites in commonly encountered in vitro biological matrices. A novel peak detection algorithm was developed to automatically detect and record the pseudomolecular ions and retention times of chromatographic components, even if not fully resolved. This algorithm was used in combination with an automated technique for predicting the molecular weights of metabolites based on incremental changes of the molecular weight of the parent drug resulting from well-known biotransformation processes. When applied to a sample of an incubation mixture of the HIV protease inhibitor Indinavir with a rat liver S9 preparation, the results obtained by the automatic metabolite detection procedures for LC-MS and LC-MS/MS analyses in real time were the same as those which were determined manually, by a knowledgeable operator.