Edward S. Riccio

The bacterial tryptophan reverse mutation assay with Escherichia coli WP2.

The Escherichia coli WP2 tryptophan reverse mutation assay detects trp(-) to trp(+) reversion at a site blocking a step in the biosynthesis of tryptophan prior to the formation of anthranilic acid. The different WP2 strains all carry the same AT base pair at the critical mutation site within the trpE gene. The assay is currently used by many laboratories in conjunction with the Ames Salmonella assay for screening chemicals for mutagenic activity. In general the WP2 strains are used as a substitute for, or as an addition to Salmonella strain TA102 which also carries an AT base pair at the mutation site. The assay is also recommended together with the Ames assay for data submission to regulatory agencies. National and international guidelines have been established for performing these mutagenicity assays. The E. coli WP2 assay procedures are the same as those described elsewhere in this volume for the Ames Salmonella assay (Mortelmans and Zeiger, 2000) with the exception that limited tryptophan instead of limited histidine is used. This chapter is an addendum to the previous chapter and the reader should refer to the previous chapter for details regarding experimental procedures and assay design.

Genotoxic properties of municipal wastewaters in Ohio

Wastewaters from six municipal wastewater treatment plants in Ohio were tested at different stages of treatment for mutagenicity in the Ames/Salmonella assay. The chlorinated secondary effluents were also evaluated for induction of sister chromatid exchanges in Chinese hamster ovary cells. Direct-acting microbial mutagenicity was observed for extracts of the effluents from all six plants for both an initial and a repeat sampling series. In some cases, the mutagenicity was greatly enhanced by S9 metabolic activation (MA). In general, the specific mutagenicity of the extracts increased following activated sludge treatment. Chlorination resulted in substantial increases in mutagenic activity for some of the Wastewaters but had no effect on others. SCE inducing activity was detected in five out of six extracts for the first sample series, and for two out of five extracts for the second sample series. There was no obvious correlation in the ability of the extracts from the chlorinated secondary effluents to induce SCE in CHO cells and to induce mutations in Salmonella.

Rat Liver Foci and in Vitro Assays to Detect Initiating and Promoting Effects of Chlorinated Ethanes and Ethylenes

Nine chlorinated aliphatics (CAs) were examined in a rat liver foci assay for tumor initiating and promoting activities. In this model, young adult male Osborne Mendel rats were first subjected to a partial hepatectomy, the test chemical was then administered at the maximum tolerated dose in the initiation or promotion phase in conjunction with diethylnitrosamine (DEN; 30 mg/kg b.w.) or phenobarbital (PB; 0.05 percent, w/w, in the diet), and gamma glutamyltranspeptidase (GGT) was used as a putative preneoplastic indicator. When administered in the promotion protocol after initiation with DEN, 1,1-dichloroethane, 1,1,2-trichloroethane (1,1,2-TCE), 1,1,2,2-tetrachloroethane (1,1,2,2-TTCE), tetrachloroethylene (TTCY), and hexachloroethane induced significant increases in GGT+-foci above control levels. 1,1,2,2-TTCE, TTCY, and 1,1,2-TCE also induced significant increases in GGT+-foci when administered in the promotion protocol without DEN initiation. Two variants of GGT+-foci were observed: the classical type associated with PB promotion, and the other, which was more diffuse, less intensely stained, resembling foci undergoing redifferentiation and associated with CAs. A number of CAs were also genotoxic in short-term in vitro tests. Taken together, the studies suggest that CAs may be complete carcinogens in vivo with weak initiating activity and stronger promoting activity.

Comparative mutagenicity of halogenated pyridines in the Salmonella typhimurium/mammalian microsome test

The Salmonella/microsome assay with strains TA97, TA98, TA100 and TA102 was used to examine the potential mutagenicity and structure-activity of 16 mono- and di-halogenated pyridines. The chemical reactivity of the halopyridines suggests that nucleophilic displacement of halogens can occur with halogens at positions 2, 4 and 6 being displaced in addition-elimination reactions. 2-Chloropyridine gave a positive result with rat-liver metabolic activation, and 2-fluoropyridine gave equivocal results under these conditions. Mutagenic responses were also obtained with 2-chloromethyl pyridine and 3-chloromethyl pyridine, in both the presence and absence of rat-liver S9. These results suggest that the halogenated pyridines, especially with halogens at the 2-position, and singly on a methyl substituent, have mutagenic activity in the Salmonella assay.

Mutagenic and carcinogenic potency of extracts of diesel and related environmental emissions: In vitro mutagenesis and DNA damage

The Saccharomyces cerevisiae D3 recombinogenic assay, the assay for forward mutagenesis in L5178Y mouse lymphoma cells, and the sister chromatid exchange (SCE) assay using Chinese hamster ovary cells were used to evaluate the in vitro mutagenic and DNA-damaging effects of eight samples of diesel engine emissions and related environmental emissions. The recombinogenic assay was not sufficiently sensitive for this evaluation, but mutagenicity was detected in the L5178Y mutagenesis assay following exposures of the cells to all of the emission samples, and DNA damage in the SCE assay was induced by most of the emission samples in the presence and absence of metabolic activation. The observation of positive results in the absence of activation indicated that the samples contained substances that were direct-acting mutagens and DNA-damaging agents.

Low-calcemic, efficacious, 1α,25-dihydroxyvitamin D3 analog QW-1624F2-2: calcemic dose–response determination, preclinical genotoxicity testing, and revision of A-ring stereochemistry

Based on an X-ray crystal structure determination, the A-ring stereochemistry of hybrid analog QW-1624F2-2 (1alpha-hydroxymethyl-16-ene-24,24-difluoro-25-hydroxy-26,27-bis-homovitamin D3) is revised to be 1alpha-CH2OH-3beta-OH. This analog is shown to be approximately 80-100 times less calciuric than the natural hormone 1alpha,25-dihydoxyvitamin D3. This analog is shown also to be non-genotoxic in three different standard assays.

Dose-dependent efficacy and safety toxicology of hydroxypyridinonate actinide decorporation agents in rodents: towards a safe and effective human dosing regimen.

Two hydroxypyridinone-containing actinide decorporation agents, 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO), are being developed for the treatment of internal actinide contamination by chelation therapy. Dose-response efficacy profiles in mice were established for the removal of intravenously injected 238Pu and 241Am after parenteral and oral treatment with these chelators. In both cases, presumed efficacious doses promoted substantially greater actinide elimination rates than the currently approved agent, diethylenetriamine-pentaacetic acid, considering two different interspecies scaling methods for the conversion of human doses to equivalent rodent dose levels. In addition, genotoxicity of both ligands was assessed using the Salmonella/Escherichia coli/microsome plate incorporation test and the Chinese hamster ovary cell chromosome aberration assay, showing that neither ligand is genotoxic, in the presence and absence of metabolic activation. Finally, maximum tolerated dose studies were performed in rats for seven consecutive daily oral administrations with the chelators, confirming the safety of the presumed efficacious doses for 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO). The results of these studies add to the growing body of evidence that both decorporation agents have remarkable decorporation efficacy properties and promising safety toxicology profiles. These results are necessary components of the regulatory approval process and will help determine the optimal human dosing regimens for the treatment of internal radionuclide contamination.

Genotoxicity and Carcinogenicity Studies of Soy Isoflavones

The potential cancer preventive efficacy of soy isoflavones is being investigated in preclinical and phase 1 clinical studies sponsored by the U.S. National Cancer Institute. Although 90-day oral toxicity studies with PTI G-2535 (an investigational soy isoflavone drug product) in rats and dogs, as well as teratology studies, indicated no signs of toxicity, there remains a mechanistic concern associated with the ability of isoflavones (i.e., genistein) to inhibit topoisomerase, possibly leading to DNA strand breaks. The present report describes results from two in vitro genotoxicity studies, one in vivo genotoxicity study, and a single carcinogenicity study conducted in p53 knockout mice. Bacterial mutagenesis experiments using six tester strains without metabolic activation revealed no evidence that PTI G-2535 was mutagenic. In similar experiments with exogenous metabolic activation there were statistically significant increases in revertants, but less than twofold, in a single (Salmonella typhimurium TA100) tester strain. Mouse lymphoma cell mutagenesis experiments conducted with and without metabolic activation revealed statistically significant increases in mutation frequency at PTI G-2535 concentrations ≥ 0.8 and 12 μg/ml, respectively; such increases were dose related and increases in the frequency of both small and large colonies were observed. A statistically significant increase in the frequency of micronucleated polychromatic erythrocytes was also seen 24 hours after treatment in male, but not female, mice who received 500 and 1000 mg/kg body weight PTI G-2535; however, such increases were small, were not dose related, and were not observed 48 hours after treatment. In contrast, dietary genistein had no effect on survival, weight gain, or the incidence or types of tumors that developed in cancer-prone rodents lacking the p53 tumor suppressor gene, p53 knockout mice. The apparent risk/benefit of isoflavone ingestion may ultimately depend on the dose and developmental timing of exposure.

Mutagenic activities of fecapentaene derivatives in the Ames/Salmonella test system

The direct mutagenic activities of a pair of naturally-occurring and several synthetic fecapentaenes were measured in the Ames/Salmonella test system. We found that strain TA100 with preincubation was the most sensitive procedure for the naturally-occurring fecapentaene-12 (FP-12). Its natural analog, FP-14, and the synthetic isomer, cis-FP-12, yielded similar mutagenic activities to FP-12 in the range of 1000-2000 TA100 revertants per microgram of compound. The synthetic analogs of FP-12 and FP-14, MFP-12 and MFP-14, wherein the glycerol moiety was replaced by methoxy, exhibited consistently higher mutagenic activities than their parent fecapentaenes (MFP-12 was about 20 times more potent than FP-12; MFP-14 was about twice the potency of FP-14). The standard rat liver metabolizing system (S9) reduced the activities of all the fecapentaenes in a dose-related manner.

Discovery and Optimization of Benzotriazine Di-N-Oxides Targeting Replicating and Nonreplicating Mycobacterium tuberculosis

Compounds bactericidal against both replicating and nonreplicating Mtb may shorten the length of TB treatment regimens by eliminating infections more rapidly. Screening of a panel of antimicrobial and anticancer drug classes that are bioreduced into cytotoxic species revealed that 1,2,4-benzotriazine di-N-oxides (BTOs) are potently bactericidal against replicating and nonreplicating Mtb. Medicinal chemistry optimization, guided by semiempirical molecular orbital calculations, identified a new lead compound (20q) from this series with an MIC of 0.31 μg/mL against H37Rv and a cytotoxicity (CC(50)) against Vero cells of 25 μg/mL. 20q also had equivalent potency against a panel of single-drug resistant strains of Mtb and remarkably selective activity for Mtb over a panel of other pathogenic bacterial strains. 20q was also negative in a L5178Y MOLY assay, indicating low potential for genetic toxicity. These data along with measurements of the physiochemical properties and pharmacokinetic profile demonstrate that BTOs have the potential to be developed into a new class of antitubercular drugs.