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The most widely used assays for detecting chemically induced gene mutations are those employing bacteria. The plate incorporation assay using various Salmonella typhimurium LT2 and E. coli WP2 strains is a short-term bacterial reverse mutation assay specifically designed to detect a wide range of chemical substances capable of causing DNA damage leading to gene mutations. The test is used worldwide as an initial screen to determine the mutagenic potential of new chemicals and drugs.The test uses several strains of S. typhimurium which carry different mutations in various genes of the histidine operon, and E. coli which carry the same AT base pair at the critical mutation site within the trpE gene. These mutations act as hot spots for mutagens that cause DNA damage via different mechanisms. When these auxotrophic bacterial strains are grown on a minimal media agar plates containing a trace of the required amino-acid (histidine or tryptophan), only those bacteria that revert to amino-acid independence (His(+) or Tryp(+)) will grow to form visible colonies. The number of spontaneously induced revertant colonies per plate is relatively constant. However, when a mutagen is added to the plate, the number of revertant colonies per plate is increased, usually in a dose-related manner.This chapter provides detailed procedures for performing the test in the presence and absence of a metabolic activation system (S9-mix), including advice on specific assay variations and any technical problems.

In vitro genotoxicity testing needs to include tests in both bacterial and mammalian cells, and be able to detect gene mutations, chromosomal damage and aneuploidy. This may be achieved by a combination of the Ames test (detects gene mutations) and the in vitro micronucleus test (MNvit), since the latter detects both chromosomal aberrations and aneuploidy. In this paper we therefore present an analysis of an existing database of rodent carcinogens and a new database of in vivo genotoxins in terms of the in vitro genotoxicity tests needed to detect their in vivo activity. Published in vitro data from at least one test system (most were from the Ames test) were available for 557 carcinogens and 405 in vivo genotoxins. Because there are fewer publications on the MNvit than for other mammalian cell tests, and because the concordance between the MNvit and the in vitro chromosomal aberration (CAvit) test is so high for clastogenic activity, positive results in the CAvit test were taken as indicative of a positive result in the MNvit where there were no, or only inadequate data for the latter. Also, because Hprt and Tk loci both detect gene-mutation activity, a positive Hprt test was taken as indicative of a mouse-lymphoma Tk assay (MLA)-positive, where there were no data for the latter. Almost all of the 962 rodent carcinogens and in vivo genotoxins were detected by an in vitro battery comprising Ames+MNvit. An additional 11 carcinogens and six in vivo genotoxins would apparently be detected by the MLA, but many of these had not been tested in the MNvit or CAvit tests. Only four chemicals emerge as potentially being more readily detected in MLA than in Ames+MNvit--benzyl acetate, toluene, morphine and thiabendazole--and none of these are convincing cases to argue for the inclusion of the MLA in addition to Ames+MNvit. Thus, there is no convincing evidence that any genotoxic rodent carcinogens or in vivo genotoxins would remain undetected in an in vitro test battery consisting of Ames+MNvit., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Dextromethorphan (DMP) is an effective and widely used antitussive drug. While DMP has over a 50 year safe-marketing history, the only available genotoxicity data was an unpublished, negative Ames assay (Roche). Lack of a complete genotoxicity profile on DMP, specifically covering the chromosomal damage endpoint, prompted a regulatory request for an in vitro chromosome aberration assay. In accordance with EC and CPMP Guidance, we evaluated data for a number of chemicals with a structural relationship to DMP. DMP contains no structural alerts for genotoxicity or carcinogenicity using the Deductive Estimation of Risk from Existing Knowledge (DEREK) software tool, confirming the negative results obtained in the existing Ames assay. This is also consistent with the mostly negative genotoxicity and carcinogenicity data available on structurally related chemicals including morphine, codeine, nalbuphine, buprenorphine, naloxone, hydromorphone, levorphanol, and oxycodone. A state-of-the-science, in vitro chromosome aberration assay was also conducted, which demonstrated a lack of genotoxicity for DMP. The overall weight of evidence for DMP and its structural analogues, supports the conclusion that this class of phenanthrene-based chemicals, and DMP, in particular, are not genotoxic in vitro or in vivo, and do not represent a carcinogenic risk to patients.

Lanthanum was evaluated for potential genotoxicity using a range of in vitro assays (as the carbonate) in the presence and absence of post-mitochondrial fraction (S9) and in vivo in three independent tests for mutagenicity and clastogenicity (as the carbonate and chloride). The drug was devoid of mutagenic activity in bacterial assays (maximum concentration 5000 microg/plate) using a range of test strains (Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100 and TA102 and Escherichia coli WP2 uvrA and WP2 uvrA pkm101). No effects were seen in the hgprt gene mutation assay in Chinese hamster ovary cells in the presence of S9. In the absence of S9, sporadic increases in revertant numbers were not dose-related or reproducible in subsequent experiments and hence were concluded to be chance events. In an in vitro chromosome aberration assay using Chinese hamster ovary cells, chromosome damage in the presence and absence of S9 (concentration 200-5000 microg/ml) was attributed to overt cell toxicity. To confirm this, a comprehensive in vivo evaluation of the drug was performed. Negative results were obtained in two independent rodent micronucleus tests. In the first mice were given oral doses (of carbonate) up to 2000 mg/kg, in the second rats were given a single i.v. bolus injection (of chloride) up to 0.1 mg/kg. Negative results were also obtained in a rat liver unscheduled DNA synthesis assay after treatment for 28 days with i.v. bolus injections (of chloride) up to 0.1 mg/kg/day. In these in vivo studies lanthanum plasma concentrations were >3000 times higher than the steady-state peak plasma concentration observed in dialysis patients given therapeutic doses of lanthanum carbonate. It can be concluded that lanthanum is not genotoxic and that lanthanum carbonate is unlikely to present a latent hazard in therapeutic use.

N-Nitrosoindoles can efficiently transfer the nitroso group to nucleophilic targets in isolated purine nucleotides, causing depurination, deamination, and the formation of a novel guanine analogue, oxanine [Lucas, L. T., Gatehouse, D., and Shuker, D. E. G. (1999) J. Biol. Chem. 274, 18319-18326]. To determine the likely biological relevance of these modification pathways, the reactivity of 1-nitrosoindole-3-acetonitrile (NIAN), a model 3-substituted N-nitrosoindole, with oligonucleotides and calf thymus DNA was examined at physiological pH and temperature. Reaction of NIAN with single-stranded oligonucleotides containing various guanine motifs resulted in the production of single-strand break products at guanine sites due to the formation of alkali-labile lesions. The number of lesions increased with NIAN concentration and incubation time. Modification of calf thymus DNA by NIAN resulted in depurination, which gave the corresponding purine bases, deamination coupled with depurination, which gave xanthine, and the formation of oxanine. The former pathway was clearly the most important, and all reaction products exhibited a dose-response relationship. Cytosine and thymine residues were inactive toward NIAN. Further studies revealed an additional product in NIAN-treated duplex DNA containing a CCGG motif that was characterized as an interstrand cross-link, the yield of which increased with increasing NIAN concentration. These results indicate that the transnitrosating ability of NIAN to modify purine residues is preserved at the macromolecular level, with guanine residues appearing to be a primary site of reaction. All of these modification processes are potentially mutagenic events if they occur in vivo.

A collaborative trial was carried out to determine the intra/interlaboratory variability of Salmonella typhimurium strains TA102 and TA97a with regard to spontaneous revertant frequency and in response to four model mutagens (cumene hydroperoxide and bleomycin for strain TA102, and 4-nitrophenylenediamine and 4-aminoantipyrine for strain TA97a). A secondary objective of the trial was to monitor the stability of the strains after storage for up to 8 months and identify any technical problems associated with their use. Thirteen different laboratories participated in the trial, all receiving identical stock cultures of the bacterial strains and samples from the same batch of mutagenic compound. A standard protocol was followed and two independent experiments were carried out within 1 month of receipt of the strains/compounds (phase I), and again after a period of 6-8 months (phase II). Comparative studies with the standard strain TA100 after treatment with 4-nitrophenylenediamine were carried out as part of phase II. Overall, both strains gave acceptably consistent results in different laboratories and are considered useful for screening purposes when used under standardized conditions. One major source of interlaboratory variability identified for TA102 appears to be the sensitivity of different types of automatic colony counter for detecting the micro-colony revertants that this strain produces.

The azo-compound, D and C Red No. 9 was assayed for genotoxicity in vivo using the rat micronucleus test and the rat ex vivo liver UDS assay. Uniformly negative results were obtained in both assays, even though large oral doses were used (2 g/kg). These results suggest that the tumorigenic effects of this compound in rats are mediated through non-genotoxic rather than a genotoxic mechanism. Further experiments using additional end-points such as 32P-post-labelling would further substantiate this conclusion.

P-Benzoquinone dioxime (BQD) appears to be a sex-specific rat carcinogen inducing tumours of the urinary bladder in female rats. The present paper shows that BQD is a direct-acting mutagen in Salmonella typhimurium TA98, confirming published data. In contrast to this in vitro data, negative results were obtained after oral administration of BQD to female rats in both the bone marrow micronucleus test and the in vivo liver UDS test. BQD did, however, induce a marked effect upon S-phase synthesis in the livers of female rats between 14 and 48 hr after a single oral dose of 250 mg/kg. A similar effect was also observed in the livers of male rats. There was no evidence of hepatotoxicity (in terms of elevated liver enzyme levels) after treatment of female rats with the compound indicating that the increase in cell proliferation was due to a direct mitogenic effect of BQD in this organ. Some liver mitogens have been found to be liver carcinogens; this does not appear to be the case for BQD. Nevertheless, the mitogenic activity of this compound might play a contributory role to the induction of bladder cancer in rats if it also acted as a mitogen in this tissue. Further studies are indicated, measuring genotoxicity and cell-proliferative activity in the bladder in order to further elucidate the mechanism of action of this compound as a rodent carcinogen.

Urethane is readily detectable as an in vivo clastogen in the micronucleus test after oral administration to both rats and mice. Mice appear to be the more sensitive species. Positive results were obtained irrespective of the strain (CD1, CRH) or dosing regimen (single, triple-dose) used.

A triple-dose protocol was evaluated in the rat bone-marrow micronucleus test using azobenzene and 1,2-dibromo-3-chloropropane (DBCP) as test compounds. Dosing with 250 mg/kg azobenzene over 3 consecutive days led to an accumulation of micronucleated polychromatic erythrocytes. The magnitude of the effect was the same as that obtained after a single dose of 750 mg/kg in a previous study. In the single-dose study the effect was only detected at the 48 h sampling time. With the triple-dose protocol the effect was apparent 24 h after the last dose--thus eliminating the need for more than one sampling time. In the case of DBCP, the positive effect observed after a single dose diminished with increasing number of doses for the low dose but plateaued for the high dose, accompanied by a marked cytotoxic effect. It is therefore concluded, that for one of the compounds, azobenzene, the triple-dose protocol provided an advantage whereas for the other, DBCP, it failed to do so and led to results which are rather difficult to interpret. Furthermore, the number of animals necessary to select an appropriate dose level appears to be higher than in the single-dose protocol.

In 1982, Levin et al. published a paper describing a new Salmonella typhimurium strain, TA102, for detecting mutagenic agents that react preferentially with AT base pairs. This strain has an AT base pair at the critical mutation site within the hisG gene, which is located on a multicopy plasmid, pAQ1; the chromosomal copy of the hisG gene has been deleted. It also has an intact excision repair system, thus facilitating the detection of cross-linking agents, and carries the mutator plasmid, pKM101. Although TA102 has been shown to be reverted by certain mutagenic agents that are not detected in the usual battery of strains (TA1535, TA1537, TA1538, TA98 and TA100), there has been a general reluctance within the field to include TA102 as one of the standard screening strains. This may in part result from the difficulties which have been experienced in many laboratories in maintaining the strain, and in obtaining reproducible spontaneous and induced revertant counts. At Glaxo we routinely include certain Escherichia coli strains in our microbial test battery, and were aware that some of the genetic features offered by TA102 were already being covered by these strains. For example, E.coli WP2 (pKM101) has an AT base pair at the critical mutation site within the trpE gene, is excision proficient (and thus will detect cross-linking agents) and carries the pKM101 plasmid to enhance error-prone repair. From the published literature it was apparent that a number of the 'TA102 specific' mutagens could be detected in E.coli e.g. neocarzinostatin, UV and 8-MOP plus UV.(ABSTRACT TRUNCATED AT 250 WORDS)

The experiments described were carried out as part of the UKEMS Third Collaborative Trial. The method used was essentially that described by Thilly et al., based on a cloning assay using microtitre test plates. The induction of mutations at both the thymidine kinase (tk) locus and the hypoxanthine guanine phosphoribosyl transferase (hprt) locus was determined following exposure to ethyl methanesulphonate (EMS), benzidine (BZD) and benzo[a]pyrene (B[a]P). EMS was tested in the absence of a metabolic activation system (S9 mix), BZD was tested in the presence and absence of S9 mix and B[a]P was tested only in the presence of S9 mix. Incubation with EMS or B[a]P caused the induction of mutations at both the tk and hprt loci. The effects of benzidine were not reproducible, although some individual data points at the tk locus were found to be significant.

The "Microtitre" Fluctuation test recently introduced for the detection of direct mutagens has been adapted for the detection of indirect mutagens through the incorporation of an "S9-mix" metabolic system. It compares favourably with Greens' original method for the detection of a range of chemical mutagens. The technique has been employed in the evaluation of mixed enzyme induction using phenobarbitone and beta-naphthoflavone (benzoflavone), as a safe substitute for Aroclor-1254. The post-mitchondrial preparations from rats induced with the combined inducers had a similar "metabolic competence" to those derived from Aroclor induced animals. Such a combination would therefore provide a useful alternative to Aroclor-1254 for routine screening. It was found that the level of "S9" present in the metabolic system greatly affected the quantitative mutagenic response. This varied considerably from chemical to chemical and underlined the need for such preliminary investigations in routine screening.

Three naturally occurring indoles were evaluated for potential nitrosatability using the Nitrosation Assay Procedure (NAP test) as recommended by the World Health Organisation. All three indoles i.e. tryptophan, tryptamine and 5-hydroxy-tryptamine were nitrosated to products which were directly mutagenic for S. typhimurium TA1537. In addition, the products of nitrosation of tryptamine and 5-hydroxytryptamine were also mutagenic for strains TA1538, TA98 and TA1535 without the need for metabolic activation. The sensitivities of the frameshift-detecting strains TA1537, TA1538 and TA98 were of particular interest, since nitroso compounds are characteristically base-substitution mutagens. The mutagenic effects of the products formed after nitrosation of each indole at pH 3.6, were eliminated in the presence of S9 mix. This was not the case when the nitrosation assay was carried out at pH 2.6. At this pH the mutagenicity of the nitrosated products varied in the presence of S9 mix and depended upon the nature of the indole undergoing nitrosation, and the bacterial test strain utilised for the mutagenicity assay. This indicated that more than one mutagenic product was responsible for the observed effects. As well as pH, a number of other factors influenced the formation of mutagenic nitroso products. Most notably, the concentrations of precursor compounds (sodium nitrite, and indole) present in the NAP test were of critical importance. As the sodium nitrite concentration was reduced from that recommended by the W.H.O. (40 mM), so the mutagenicity decreased. For all three compounds significant mutagenic effects were lost at sodium nitrite concentrations below 15 mM. In conclusion the data presented in this paper clearly demonstrates that individuals are chronically exposed to naturally occurring substances which readily nitrosate in excess nitrous acid and yield bacterial mutagens.

The World Health Organisation (WHO) has recently recommended the use of a simple in vitro test (Nitrosation Assay Procedure, NAP) to allow the ranking of orally administered drugs. It was suggested that the ranking of drugs should be carried out on a reactivity scale, according to their ability to nitrosate in the presence of high concentrations of nitrite at low pH (3-4). The present study was undertaken to investigate the effect of adding the concentration of nitrite recommended for the NAP Test (40 mM) to normal fasting human gastric juice. The data clearly demonstrate that such treatment results in the formation of derivatives with specific mutagenic activity for the bacterium Salmonella typhimurium TA1537. This strain was recommended for use in the NAP Test. Although the NAP Test may be valid for its original purpose, in the absence of more physiologically relevant data it is invalid for use as a final arbiter in risk-benefit assessments. The possible formation of mutagenic species as a result of the nitrosation of natural gastric secretions should be considered when any risk-benefit assessment is made.

Loxtidine, a potent, non-competitive histamine H2-receptor antagonist was evaluated for genotoxic potential using a range of short-term mutagenicity assays. Unequivocally negative results were obtained in a Salmonella/plate incorporation assay and a liquid pre-incubation assay (using S. typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98), a fluctuation assay [using Escherichia coli strains WP2, WP2 uvrA (R46) and 343/113 lys60 (R46)], a gene conversion assay (using Saccharomyces cerevisiae JD1) and a human peripheral lymphocyte cytogenetic assay. All of these in vitro tests were carried out in the presence and absence of rat liver S9 mix. In addition, the major metabolites of loxtidine in the rat were also negative in the same range of microbial mutagenicity assays. Loxtidine was inactive in the mouse micronucleus test after oral administration. The potential nitrosatability of loxtidine was investigated using an expanded version of the WHO Nitrosation Assay Procedure, and detectable quantities of mutagenic nitroso-species were not formed. The subsequent appearance of carcinoid tumours within the gastric fundus of rodents treated orally with loxtidine for most of their natural lifespan, led to additional assays being carried out on this compound to determine whether the tumorigenic effects were due to alternative mutagenic mechanisms. Negative results were obtained in an in vitro unscheduled DNA synthesis assay using primary rat hepatocytes, and an assay for spindle damaging agents using Muntjac skin fibroblasts. It can be concluded from these results that loxtidine is unlikely to be a genotoxic carcinogen. The increase in carcinoid tumour incidence observed in rats and mice after loxtidine treatment was probably related to the prolonged achlorhydria produced by this potent unsurmountable histamine H2-receptor antagonist.

The anti-tuberculostatic drug, isoniazid (INH) was evaluated for its mutagenic potential using Salmonella plate tests and fluctuation assays with various strains of bacteria, and different metabolic activation systems. In the Salmonella plate test INH proved to be a weak directly-acting base-substitution mutagen which was detoxified by S9-mix. S. typhimurium TA 1530 and TA 1535 were the sensitive strains, and this result confirmed some of the published data. In the present studies mutagenic activity was further diminished in the presence of larger concentrations of rat liver S9-mix. Furthermore, the reduction in mutagenic activity was observed with S9-mix derived from untreated, Aroclor 1254-treated or phenobarbitone/beta-naphthoflavone treated rats. In direct contrast, using the microtitre fluctuation assay, the mutagenic activity of INH was elevated in the presence of rat liver S9-mix, and continued to increase with increasing S9-concentration. This result was obtained irrespective of the S9-source. S. typhimurium strains TA 1530, TA 1535 and his G46, and E. coli strains TA 85, TA 86 and WP2 uvrA were all sensitive to the mutagenicity of INH after metabolic activation. The primary step in the metabolic activation of INH in the fluctuation test was mediated by a cytosolic enzyme, and the activity of dapsone as a competitive substrate implicated the involvement of an N-acetyl transferase. The rapid diffusion of the cytosolic enzyme into the basal agar layer, or the non-specific binding of the enzyme (or the active mutagenic INH metabolite) to components of the agar, may explain the contradictory data obtained in the Salmonella plate test. The modifying effects of agar on the distribution of drug metabolising enzymes within liver S9 fractions should be carefully considered when evaluating data from Salmonella plate tests.

2-Nitropropane (2-NP) is a rat liver carcinogen, whilst the 1-isomer is non-carcinogenic in rodents. Although DNA repair tests in the rat liver discriminated clearly between the carcinogenic and the non-carcinogenic isomer, uniformly negative results have been published for the mouse bone marrow micronucleus test (BMMN test) with both isomers. Therefore, the latter assay did not discriminate between the carcinogenic and the non-carcinogenic isomer. To investigate whether this is due to endpoint specificity or organospecificity of 2-NP, studies were carried out in the rat in which micronucleus induction (bone marrow and liver) and unscheduled DNA synthesis (UDS) induction (liver) were measured after oral treatment with either nitropropane isomer. 2-NP induced UDS in the liver whilst the 1-isomer was negative, thus confirming the published studies. In the BMMN test, occasional small increases in the incidence of micronuclei were found for both compounds, but results were interpreted as negative after considering the control background data and the lack of reproducibility. By contrast, the liver micronucleus test revealed a clastogenic effect of 2-NP in the liver. This indicates that 2-NP induces chromosome aberrations as well as DNA repair in vivo, but it seems to act organospecifically. For 1-NP a slightly increased incidence of micronuclei was found in the liver, which was accompanied by a markedly increased mitotic index. It therefore remains questionable as to whether this increased micronucleus frequency for 1-NP is an indicator of a clastogenic effect, or whether it is caused by an increased cell proliferation induced by 1-NP. Consequently, it is too early to conclude whether the liver micronucleus assay is able to discriminate between the carcinogenic and non-carcinogenic isomer. However, the results provide further evidence that bone marrow assays are insufficient for the detection of all genotoxic carcinogens in vivo. This indicates the need for analysing a second tissue, particularly when negative bone marrow results have been obtained with in vitro genotoxins.

This review discusses features of the standard protocols currently used in bacterial mutation assays which can have a critical effect upon the test outcome. Such features if not strictly controlled may affect the results qualitatively as well as quantitatively. These include the dose-intervals of the test compound used; the number of bacterial cells exposed to the test compound; the phase of growth such cells are in during exposure; and the length of time that the cells are exposed prior to the addition of soft agar (in the Salmonella/plate incorporation assay). The following possible modulating effects will also be discussed: the nature of the solvent used; the nature and quantity of the exogenous metabolizing system (usually liver S9-fraction) and the quantity of amino-acid (e.g. histidine) within the test system being that either deliberately added or present unavoidably within the test sample (e.g. biological fluids). If bacterial mutagenicity assays are to realize their full potential for the detection of genotoxic carcinogens, the use of rigid protocols should be discouraged. Where possible, consideration of the compound's structure should lead to the employment of the optimal protocol for the detection of genotoxic carcinogens within that chemical class. The relative speed and low cost of bacterial assays should be exploited in this way to avoid the generation of false negative results during the primary screening of novel compounds.

A number of 1,2-ring fused acenaphthenes, together with the parent compounds acenaphthene and acenaphthylene, were evaluated for mutagenicity, using the Pour-Plate Technique with S. tpyhimurium strains TA1538 and TA1537. Although acenaphthene and acenaphthylene were non-mutagenic, all the 1,2-ring fused acenaphthene were found to be indirect frameshift mutagens in strain TA1537. The chemical nature of the 1,2-fused ring did not appear to be important for mutagenic activity against TA1537, however, its nature did affect the mutagenesis of strain TA1538. Only acenaphthenes fused with a pyrimidine or pyrazine ring were capable of mutating the hiD 3052 locus of TA1538. Substitution at the 8-position of the ring system with an amino group rendered the molecule inactive against TA1538, whilst substitution at the 10-position only reduced, but did not eliminate the mutagenic effect against TA1538. Methyl substitution at various sites on the molecule modified the mutagenic activity against TA1537, and indicated the formation of an electrophilic species (epoxide) at the 2,3-position of the acenaphthene nucleus. The incorporation of a competitive substrate for ring hydroxylation (naphthalene) reduced the mutagenic effect of acenaphthopyrimidine against TA1537 and confirmed this assumption. However, naphthalene did not reduce the mutagenic effect of the compound against TA1538, indicating the possible formation of a second metabolite by an alternative enzymic pathway. The fusion of a pyridine ring to the system to give a pentacyclic compound resulted in a molecule sufficiently planar to allow for a weak direct mutagenic effect against TA1537.

The anti-neoplastic agent procarbazine is genetically active in a variety of short term mutagenicity tests, and it also possesses carcinogenic and teratogenic potential. This compound has consistently yielded false negative results in in vitro microbial mutagenicity tests in the presence and absence of mammalian metabolic activation. In this study, procarbazine was not mutagenic in standard and preincubation Ames tests using large S9 concentrations and bacterial test strains devoid of the rfa mutation. The microtitre fluctuation test is a sensitive technique for the detection of bacterial mutagens. Using this assay, procarbazine proved to be a bacterial mutagen after in vitro metabolic activation at concentrations as low as 200 micrograms/ml. Activity was dependent upon liver S9-concentrations which were higher than those usually attained within agar assays, and was only observed when such fractions were derived from aroclor-treated or phenobarbitone plus beta-naphthoflavone treated rat livers. The excision-repair proficient strain E. coli WP2 was equally, if not more, sensitive than the repair deficient strain E. coli WP2 uvrA. Furthermore, the differential mutagenic effects obtained using S. typhimurium strains TA1535, TA1530 and his G46 indicated that the absence of an rfa mutation was essential for procarbazine mutagenesis. Procarbazine was also mutagenic for E. coli D494 in a forward mutation fluctuation assay measuring resistance to ampicillin. In this assay lower concentrations of S9-fraction were sufficient, reflecting the increased sensitivity of the test strain towards the mutagenic metabolites of the drug. In conclusion, the results suggest that rat liver S9-fraction contains only low levels of enzymes capable of activating procarbazine to a mutagen. This may be a result of rapid breakdown during storage, or due to intrinsically low levels in rat liver extracts. Inducing agents appear to increase these levels. The low concentrations of mutagenic species formed in vitro, may only be detectable using a highly sensitive assay such as the microtitre fluctuation test.

The in vitro and in vivo clastogenic potential of three beta-lactam antibiotics, ampicillin, carbenicillin and penicillin VK, was investigated using cultured human lymphocytes and the rat micronucleus test. Neither ampicillin nor carbenicillin induced significant increases in chromosome damage in vitro up to test concentrations of 10 mg/ml. These results contrast with other published studies on these compounds. Both drugs were also inactive in vivo in the rat micronucleus test, using single- or double-dosing regimens (ampicillin 5 g/kg orally; carbenicillin 500 mg/kg i.m., either dosed once 30 h before marrow preparation, or dosed twice 48 and 24 h before marrow preparation). In vitro, penicillin VK induced a dose-related increase in chromosome and chromatid gaps and breaks, down to concentrations of 1.25 mg/ml. It is likely that the increase in aberration frequency was partly the result of exposing the cells to increased K+ ion concentration, as similar results were obtained when potassium chloride was evaluated over the same molar concentration range. However, the occurrence of 'ion-mediated' clastogenic effects as reported by other workers, does not fully account for the positive effects obtained with this compound, as clastogenic effects were also observed with penicillin V in this test system at similar test concentrations. It is known that exposure of mammalian cells to extremely high concentrations of beta-lactams can affect DNA polymerase alpha activity. An inhibitory effect upon DNA polymerase alpha resulting in a breakdown in the structural integrity of the chromosomes, is suggested as an additional mechanism of action for penicillin VK.(ABSTRACT TRUNCATED AT 250 WORDS)