Comparative mutagenicity testing of bropirimine, 1. Induction of chromosome aberrations in CHO cells is not reflected in induction of mutation at the TK locus of L5178Y cells

Bropirimine (U-54,461) is a novel compound which is being developed as a biological response modifier for use in treatment of neoplastic and viral disease. Compounds of this type exert their therapeutic effects by immuno-stimulation or other non-cytotoxic mechanisms. The purpose of the experiments described in this paper was to evaluate the hazard potential of this drug. Bropirimine was previously reported to be negative in the Ames Salmonella assay (Aaron et al., 1989a) and the in vitro UDS assay (Aaron et al., 1989b). In experiments reported here positive response was observed in a test for clastogenicity in vitro in CHO cells, but bropirimine was negative in the L5178Y mouse lymphoma TK^+/− assay. A subsequent experiment demonstrated the ability of bropirimine to induce HPRT mutations in CHO cells. Interestingly, evidence for induction of chromosome aberrations in the L5178Y cells by bropirimine was also obtained. While the reason for the apparent insensitivity of the L5178Y TK^+/− assay to bropirimine is unexplained by the experiments, it is clear that at high dose bropirimine is capable of clastogenesis in both CHO and L5178Y cells and can give rise to gene mutations in CHO cells but apparently not in L5178Y cells.     

Carcinogenicity and mutagenicity testing, then and now.

Cancer is a dread disease worldwide. Mortality of individuals suffering from cancer is high, despite the current improved methods of precocious detection, surgery and therapy. Prevention of cancer is the recognized goal of many activities in cancer research. This aim was recognized early to involve the bioassay of environmental chemicals or mixtures. The first such study involved application of coal tar to the ear of rabbits, and later on to the skin of mice. Subsequently, laboratory rats were introduced, and hamsters were utilized as a substitute for the unwieldy tests in rabbits. Investigators also became concerned with the mechanisms of carcinogenesis, and more definitive approaches to carcinogen bioassay in laboratory animals, as possible indicators of cancer risk in humans. These tests were expensive and lengthy, and did not serve the important purpose of accurately measuring risk of cancer to humans. Once it was realized that DNA and the genetic apparatus might be a key target, rapid bioassays in bacterial and mammalian cell systems were introduced successfully. Thus, batteries of tests are now available to detect effectively human cancer risks, and provide novel approaches to determine the underlying mechanisms, as a sound basis for cancer prevention.     

Legislative and technical aspects of mutagenicity testing.

A brief account is given of the history of the legislative acts that give responsibility to the U.S. Food and Drug Administration (FDA) for ensuring the safety of foods, drugs, and cosmetics. Within the present legislative framework the FDA has the authority to impose regulations which are designed to ensure the safety of all foods, drugs, and cosmetics. The existing legislative authority is adequate for this purpose; however, the difficulty lies instead with technology and the inadequacy of scientific perspective in the emerging area of mutagenicity testing. Earlier efforts in development of mutagenicity screening systems culminated only a few years ago in the proposal to use the host-mediated assay, somatic cell cytogenetics, and dominant lethal tests collectively. Subsequent research efforts indicated that there were serious practical and scientific deficiencies in using this approach. More recently a new proposal, the tier system, has been suggested as an alternative measure. The proposed tier system at FDA consists of three testing levels of increasing complexity. The first tier is an initial screening effort using techniques having maximum sensitivity that are also useful for large-scale, rapid testing. The second tier is designed to identify and confirm that the presumptive mutagens detected in the first tier are truly mutagenic for higher organisms, most especially, for mammals. The third tier would be devoted to explicit genetic tests in mammals designed to ascertain the imposed risk to man by the introduction of a mutagen in our environment. The FDA is currently involved in a number of research activities in the area of mutagenicity safety screening which will explore the adequacies and possible deficiencies of the tier system approach. These efforts are described for our in-house activities, our contract activities, and our cooperative and collaborative activities with other government agencies and institutions.     

delta 1-Tetrahydrocannabinol and 1 alpha, 2 alpha-epoxyhexahydrocannabinol: mutagenicity investigation in the Ames test.

1,2-Epoxyhexahydrocannabinol is a metabolite of delta 1-tetrahydrocannabinol. Because many epoxides are mutagens, we investigated 1,2-epoxyhexahydrocannabinol as well as delta 1-tetrahydrocannabinol for mutagenicity with Salmonella typhimurium TA1535, TA1537, TA98 and TA100 in the presence and in the absence of S9 mix from liver homogenate of rats treated with Aroclor 1254. Additionally, an epoxide hydratase inhibitor was used in some experiments. Whereas several other epoxides and further positive controls, not requiring activation or activated under the same conditions, respectively, showed strong mutagenicity, no indications of a mutagenic hazard by 1,2-epoxyhexahydrocannabinol or by delta 1-tetrahydrocannabinol were found.     

Comparative in vivo mutagenicity testing by SCE and micronucleus induction in mouse bone marrow

Summary The treatment of mice with repeated injections of BUdR and FUdR allows for the demonstration of differentially stained metaphases from bone marrow after FPG (fluorescence plus Giemsa; Perry and Wolff, 1974) treatment. Thus, it is possible to determine the number of SCE's under in vivo conditions, which appears as a very promising system for mutagenicity testing. We studied the response of this system in comparison to the micronucleus test using six mutagenic agents: triaziquone, cyclophosphamide (CP), dimethylphenyltriazene (PDMT), methylnitronitrosoguandine (MNNG), dimethylnitrosamine (DMNA), and diethylnitrosamine (DENA). With the exception of MNNG and DENA, all these agents induce both, SCE and micronuclei, MNNG and DENA being ineffective in both systems. The most potent SCE-inducing agent was triaziquone, followed by PDMT, CP, and DMNA. The quantitative comparison indicates that SCE are induced at 1/10–1/100 of the concentrations which are required for the detection of micronuclei.     

Comparative toxicity and mutagenicity of N-hydroxy-2-acetylaminofluorene and 7-acetyl-N-hydroxy-2-acetylaminofluorene in human lymphoblasts.

Exponentially growing TK6 human lymphoblasts were exposed to either 0-50 microM N-hydroxy-2-acetylaminofluorene (N-OH-AAF) or 0-10 microM 7-acetyl-N-hydroxy-2-acetylaminofluorene (7-acetyl-N-OH-AAF) in both the absence and presence of a partially purified preparation of hamster-liver N-arylhydroxamic acid N,O-acyltransferase (AHAT). Neither N-arylhydroxamic acid was toxic to the lymphoblasts, nor mutagenic at the thymidine kinase (tk) locus, in the absence of AHAT over the concentration range examined. In the presence of AHAT, an enzyme that activates N-arylhydroxamic acids to electrophilic N-acetoxyarylamine intermediates, both compounds caused toxicity and mutagenicity in TK6 cells. The 7-acetyl-N-OH-AAF was approximately 10-fold more toxic and mutagenic than the unsubstituted N-OH-AAF. These data demonstrate that metabolism of these N-arylhydroxamic acids, presumably to N-acetoxyarylamine intermediates by AHAT, is a key event in the biological activity of these agents. In addition, the presence of electron-withdrawing 7-acetyl substituent that is thought to stabilize N-acetoxy intermediates, appears to enhance the biological activity of the unsubstituted N-OH-AAF.     

Lipocalin-2: development, analytical characterization, and clinical testing of a new ELISA.

Lipocalin-2 (also known as neutrophil gelatinase-associated lipocalin [NGAL]) has been described as a promising marker of metabolic syndrome associated with inflammation. The aim of our work was to develop an assay for the determination of lipocalin-2 in human serum and to investigate its levels in healthy volunteers and donors suffering from metabolic syndrome. We also conducted a pilot study on individuals with metabolic syndrome and on healthy probands and measured lipocalin-2 in these individuals. We developed and evaluated the sandwich ELISA method for the quantitative determination of human lipocalin-2 in serum samples. We measured blood pressure, waist circumference, serum cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, insulin, glucose, creatinine, hs-CRP, and adiponectin and calculated the BMI and Quicki insulin sensitivity index. In the study on 153 healthy volunteers, we showed that sex and age are not determinative for lipocalin-2 serum values. Furthermore, we tested 45 individuals with metabolic syndrome; values of lipocalin-2 did not differ (78.8 vs. 80.0 microg/l, p =0.56) from the data of healthy individuals from the first study. Neither group differed with regard to sex or age. Lipocalin-2 correlated with alanine aminotransferase (ALT) (r=-0.3, p<0.01) aspartate aminotransferase (AST) (r=-0.3, p<0.01), cholesterol (r=-0.21, p=0.047), creatinine (r=0.19, p=0.05), and high-sensitivity C-reactive protein (hs-CRP) (r=0.22, p=0.036). No significant correlation was found between serum lipocalin-2 and BMI, waist circumference, blood pressure, triglycerides, HDL, Quicki, or the number of metabolic syndrome components. When study patients with metabolic syndrome were further stratified according to the number of components of metabolic syndrome, serum concentrations of lipocalin-2 did not differ. The results presented demonstrate the analytical competence of the lipocalin-2 assay. However, we assumed that lipocalin-2 is not a routinely usable marker of metabolic syndrome or obesity. The association between serum lipocalin-2 and obesity or metabolic syndrome was not validated in our study.     

Bacterial mutagenicity investigation of epoxides: drugs,drug metabolites,steroids and pesticides

Although it has been observed that many epoxides are ultimate mutagens, surprisingly little is known about epoxides to which man may be extensively exposed, e.g., physiological compounds, drugs, drug metabolites and pesticides. We have now investigated 35 such and related epoxides for mutagenicity, using reversion of his^?Salmonella typhimurium TA98 and TA100 as biological end-point. None of the tested steroids (12 compounds), vitamin K epoxides (3 compounds) and pesticides (dieldrin, endrin, HEOM (1,2,3,4,9,9-hexachloro-6,7-epoxy-1,4,4a5,6,7,8,8a-octahydro-1,4-methanonaphthalene), heptachlor epoxide) showed any mutagenic activity. Negative results were also obtained with the antibiotics oleandomycin, anti-capsin and asperlin, the cardiotonic drug resibufogenin, the widely used parasympatholytic drugs butylscopolamine and scopolamine, the sedatives valtratum, didovaltratum and acevaltratum, the tranquilizer oxanamide as well as with the drug metabolites carbamazepine 10,11-oxide and diethylstilbestrol α,β-oxide. Three barbiturate epoxides, formed by metabolism of allobarbital, alphenal and secobarbital, caused weak but reproducible mutagenic effects at high concentrations. The cytostatic agent ethoglucide was the only drug having substantial mutagenic activity. Its mutagenic potency was similar to those of the control epoxides styrene 7,8-oxide, p-bromostyrene 7,8-oxide and m-bromostyrene 7,8-oxide, but much lower than those of benzo[a]pyrene 4,5-oxide, benzo[e]pyrene 4,5-oxide and 7,12-dimethylbenz[a]-anthracene 5,6-oxide.Some epoxides were also tested in other Salmonella typhimurium strains or in the presence of rat-liver S9 mix. Positive results were only obtained with compounds that had already been detected as mutagens in the direct test with strain TA100.     

Further characterization of the covalent linking reaction of alpha 2-macroglobulin.

It is shown that non-proteolytic proteins can become covalently linked to alpha 2M (alpha 2-macroglobulin) during its reaction with proteinases, and that this probably occurs by the mechanism that leads to the covalent linking of proteinases described previously [Salvesen & Barrett (1980) Biochem. J. 187, 695-701]. The covalent linking of trypsin was at least partly dependent on the presence of unblocked lysine side chains on the protein. The covalent linking of proteinases was inhibited by nucleophiles of low Mr, and these compounds were themselves linked to alpha 2M in a molar ratio approaching one per quarter subunit. Peptide "mapping" indicated that the site of proteinase-mediated incorporation of the amines was the same as that at which methylamine is incorporated in the absence of a proteinase. The nucleophile-reactive site revealed in alpha 2M after reaction with a proteinase was shown to decay with a t1/2 of 112 s, at pH 7.5. After the reaction with a proteinase or with methylamine, a free thiol group was detectable on each subunit of alpha 2M. We propose that the site for incorporation of methylamine in each subunit is a thiol ester, which in S-alpha 2M (the electrophoretically "slow" form) is sterically shielded from reaction with large nucleophiles, but is revealed as a highly reactive group, free from steric hindrance, after the proteolytic cleavage. We have designated the activated species of the molecule "alpha 2M".     

Comparative mutagenicity of ICR-191 to S. typhimurium and diploid human lymphoblasts.

Concentration-dependent mutagenicity of ICR-191 has been measured in Salmonella typhimurium strain TA98 and in a diploid human cell line. In both cell systems, approximately equigenerational exposure produced mutation linearly related to concentration in the lower range of ICR-191 concentrations tested. Saturation behavior was observed in the human cell assay but not in the bacterial assay. However, a 25-fold greater concentration of ICR-191 was required to induce a significant rise in the mutant fraction in the S. typhimurium assay than in the human cell assay. These differences may be linked to the differences in the biochemical events required for mutation or in the time of exposure to ICR-191.     

Further characterization of bovine pancreatic lipase.

1. The amino acid composition of bovine pancreatic lipase is very similar to that of porcine lipase. 2. Bovine lipase possesses a residue of lysine at the N-terminal position and a half cystine or a cysteine at the C-terminal position. 3. Bovine lipase contains two free sulfhydryl groups of different reactivities to 5, 5'-dithiobis-(2-nitrobenzoic) acid. One of these groups is buried in the native conformation of the enzyme and is fully titrated in 1.5 M urea when reaction is performed in the presense of 1 mM EDTA.     

Further characterization of mitochondrial protein phosphatase.

Mitochondrial protein phosphatase from rat liver exhibits rather wide substrate specificity, since it readily dephosphorylates, besides phosvitin, casein, and cytosol phosphoproteins, also ATP, ADP, inorganic pyrophosphate, p-nitrophenylphosphate.Aliphatic phosphate esters (β-glycerophosphate, glucose-6-P, serine-phosphate) are not dephosphorylated to any detectable extent.Evidence for the participation of a single enzyme in the dephosphorylation of phosvitin and ATP is provided. However, the different affinity toward the two substrates and other evidence suggest that the enzyme has in vivo the biological role of dephosphorylating, at least preferentially, the phosphoproteins.     

Further characterization of human eosinophil peroxidase.

The large and the small subunits (Mr 50 000 and 10 500 respectively) of human eosinophil peroxidase were isolated by gel filtration under reducing conditions. The subunits were very strongly associated but not apparently cross-linked by disulphide bridges. During storage, the large subunit tended to form aggregates, which required reduction to dissociate them. Amino acid analysis of the performic acid-treated large subunit showed the presence of 19 cysteic acid residues. The small subunit of eosinophil peroxidase had the same Mr value as the small subunit of myeloperoxidase. However, although these subunits have very similar amino acid compositions, they showed different patterns of peptide fragmentation after CNBr treatment. The carbohydrate of eosinophil peroxidase seemed associated exclusively with the large subunit and comprised mannose (4.5%, w/w) and N-acetylglucosamine (0.8%, w/w). The far-u.v.c.d. spectrum of the enzyme indicated the presence of relatively little ordered secondary structure.     

The use of transgenic mice for short-term,in vivo mutagenicity testing

In order to develop a short-term, in vivo assay to study the mutagenic effects of chemical exposure, transgenic mice were generated using a lambda shuttle vector containing a lacZ target gene. Following exposure to mutagens, this target can be rescued efficiently from genomic DNA prepared from tissues of the treated mice using restriction minus, in vitro lambda phage packaging extract and restriction minus Escherichia coli plating cultures. Mutations in the target gene appear as colorless plaques on a background of blue plaques when plated on indicator agar. Spontaneous background levels were ′1 × 10⁻⁵ in each of three mouse lineages analyzed. Exposure of lambda transgenic mice to N-ethyl-N-nitrosourea resulted in as much as a 14-fold induction in detected mutations over background levels. The assay is currently being modified to incorporate lacI as the target for ease of mutation detection as well as in vivo excision properties of the Lambda ZAP vector, facilitating sequence analysis of mutant plaques.     

Comparative mutagenicity of alkylsulfate and alkanesulfonate derivatives in Chinese hamster ovary cells.

Mutation induction and cell killing produced by selected alkylsulfates and alkanesulfonates have been quantitated using the Chinese hamster ovary/hypoxanthine--guanine phosphoribosyl transferase (CHO/HGPRT) system. Dose--response relationships of cytotoxicity and mutagenicity are presented for two alkylsulfates [dimethylsulfate (DMS), diethylsulfate (DES)] and three alkyl alkanesulfonates [methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), and isopropyl methanesulfonate (iPMS)]. Under the experimental conditions employed, cytotoxicity decreased with the size of the alkyl group. DMS was more toxic than DES, and MMS was more toxic than EMS and iPMS. All agents produced linear dose--response of mutation induction: DMS was more mutagenic than DES, and MMS was more mutagenic than EMS and iPMS based on mutants induced per unit mutagen concentration. However, the following relative mutagenic potency was observed when comparisons were made at 10% survival: DES greater than DMS; EMS greater than MMS greater than iPMS.