Correlation between in vivo and in vitro toxicity of some chlorinated aliphatics.

The release of lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) from rat and mouse hepatocytes was used to rank the in vitro cytotoxic potential of eight chlorinated aliphatic compounds. The rankings were compared with toxicity rankings based on the maximum tolerated doses for each species. Significant correlations between the rankings were obtained when air: water partition coefficients were factored into the in vitro data (EC(50)s) in order to adjust for differences in volatility and the retention of the compounds in vivo. For both rats and mice, as an indicator of cytotoxic potency, LDH release correlated better with hydrocarbon toxicity than AST release did. These data support the use of isolated hepatocytes for screening of chemicals for relative toxicity.

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.

Differences in rat liver enzyme-altered foci produced by chlorinated aliphatics and phenobarbital.

Nine chlorinated aliphatics (CAs)--1,1-dichloroethane, 1,2-dichloroethane, 1,1,1-trichloroethane, 1,1,2-trichloroethane, trichloroethylene, tetrachloroethylene, 1,1,1,2-tetrachloroethane, 1,1,2,2-tetrachloroethane, and hexachloroethane--were examined in a rat liver foci assay for evidence of initiating and promoting potential. Young adult male Osborne-Mendel rats (ten/group) were given partial hepatectomies, followed 24 hr later by a single i.p. dose of either diethylnitrosamine (30 mg/kg body weight) or CA, 1 wk later either a diet containing 0.05% (w/w) phenobarbital or daily oral gavage (5 X/wk) of CA in corn oil for 7 weeks, and sacrificed 1 wk later. Putative preneoplastic markers monitored were foci with increased gamma-glutamyltranspeptidase activity [GGT(+)]. CAs were without significant effect in the initiation protocol at the maximum tolerated dose. In the promotion protocol, 1,1-dichloroethane, 1,1,2-trichloroethane, tetrachloroethylene, 1,1,2,2-tetrachloroethane, and hexachloroethane induced significant increases in GGT(+) foci above control levels. Two variants of GGT(+) foci were distinguishable, one associated predominantly with phenobarbital promotion, resembling preneoplastic foci in other models, and the other associated with CA promotion, which was less intensely stained and exhibited branching, resembling foci undergoing redifferentiation. The marked differences in response may relate to differences in cytotoxic potential or mechanism of action of the two types of agents.

In vitro transformation of BALB/c-3T3 cells by chlorinated ethanes and ethylenes.

Eight chlorinated ethanes and 3 chlorinated ethylenes were tested in the BALB/c-3T3 cell transformation assay. Under the conditions of the assay, vinyl chloride and 1,1,1-trichloroethane induced a clear positive transformation response while 1,1,2-trichloroethane and trichloroethylene were weakly positive. Chloroethane, 1,1- and 1,2-dichloroethane, 1,1,1,2- and 1,1,2,2-tetrachloroethane, hexachloroethane and tetrachloroethylene were all negative in the assay conducted in the absence of an exogenous metabolic activation system. These results suggest that the BALB/c-3T3 cells possess capability to activate some, but not all, of the chlorinated hydrocarbons which exhibit species specificity in producing carcinogenicity in mice but not in rats.

Metabolic disposition study of chlorinated hydrocarbons in rats and mice.

Chlorinated hydrocarbons found in a bioassay to be carcinogenic to both B6C3F1 mice and Osborne-Mendel rats (1,2-dichloroethane), carcinogenic only to mice (1,1,2-trichloroethane, 1,1,2,2-tetrachloroethane, hexachloroethane, trichloroethylene, and tetrachloroethylene), and noncarcinogenic to either species (1,1-dichloroethane and 1,1,1-trichloroethane) were used to investigate the biochemical bases for tumorigenesis. Studies were conducted after chronic oral dosing of adult mice and rats with the MTD and 1/4 MTD of each compound. The extent to which the compounds were metabolized in 48 hr, hepatic protein binding, and urinary metabolite patterns were examined. Metabolism of the compounds (mmoles per kg body weight) was 1.7 to 10 times greater in mice than in rats. Hepatic protein binding (nanomole equivalents bound to 1 mg of liver protein) was 1.2 to 8.3 times higher in mice than in rats except for 1,2-dichloroethane and 1,1,1-trichloroethane. The noncarcinogens 1,1-dichloroethane and 1,1,1-trichloroethane exhibited 2 to 18 times more binding in mice than did the carcinogens 1,2-dichloroethane and 1,1,2-trichloroethane. Urinary metabolite patterns of the compounds were similar in both species. The biochemical parameters measured provided no clue to differentiate the carcinogens from the noncarcinogens.

Apparent correlation between structure and carcinogenicity of phenylenediamines and related compounds.

The carcinogenicity of 23 phenylenediamines and related compounds was reviewed. An extensive analysis of the methods used indicated that the bioassays were conducted well. The data suggest that the carcinogenicity of 4-substituted 1,3-phenylenediamines is reduced substantially or eliminated completely by oxidation of one or both amine groups or by N-substitution. Oxidation of a methyl substituent on nitroaniline to a carboxyl group eliminated all carcinogenic activity. It required dichlorination to make ring-substtuted 1,4-phenylenediamine carcinogenic whereas only one chlorine atom was needed to make 1,2- and 1,3-phenylenediamine carcinogenic. While the available data suggest that as a class, 4-substituted 1,3-phenylenediamines are carcinogenic more often than ring-substituted 1,4-phenylenediamines, the type of added substituent and its position on the benzene ring also are important in exerting carcinogenic activity.

An overview of current efforts in short-term carcinogen testing.

Scientists in the Health and Environmental Review Division (HERD), Office of Toxic Substances of the U.S. Environmental Protection Agency, are examining the feasibility of expanding efforts in short-term carcinogen testing. Three areas for consideration have been defined. These are (1) short-term in vitro tests; (2) short-term in vivo tests; and (3) tumor markers. HERD's current efforts in short-term in vitro testing are exemplified by the Gene-Tox program. Through a comprehensive system of committees and reviews, the published literature on eukaryotic and prokaryotic in vitro and in vivo test systems are being examined and analyzed. The suitability of utilizing the various systems in a test battery to identify potential chemical mutagens and carcinogens will be ascertained. A review of the literature on short-term in vivo tests (limited bioassays) and tumor markers is currently being conducted. Correlations will be made between results obtained from these tests and epidemiological information and long-term animal bioassays. The attributes and deficiencies of each test or marker will be examined. Further testing, development, or validation needs will be outlined. The aim of this review is to attempt to expand the prechronic test battery for carcinogenicity in order to provide sufficient information for regulatory decision-making.

Review of experimental carcinogenesis by compounds related to vinyl chloride.

The experimental carcinogenesis results in six compounds related to vinyl chloride are reported. Vinylidene chloride, given by inhalation, was carcinogenic in male CD-1 mice, male CD rats, Sprague-Dawley rats and male Swiss mice. Trichloroethylene, given by gavage and inhalation, was carcinogenic in the B6C3F1 mice. When given by gavage, perchloroethylene was carcinogenic in the B6C3F1 mice, and dichloroethane was carcinogenic in Osborne-Mendel rats and B6C3F1 mice. Dibromoethane, given by gavage and inhalation, was carcinogenic in B6C3F1 mice, F344 rats and Osborne-Mendel rats. Finally, epichlorohydrin was carcinogenic in male Sprague-Dawley rats and B6C3F1 mice.

Studies on the mechanism of the glutamine-dependent reaction catalyzed by asparagine synthetase from mouse pancreas.

Initial velocity and product inhibition studies were conducted with the glutamine-dependent reaction of asparagine synthetase from mouse pancreas. Double reciprocal plots of glutamine versus either aspartate or ATP were parallel, while aspartate versus ATP gave intersecting patterns. These patterns are indicative of a hybrid ping-pong mechanism consisting of a glutaminase partial reaction and a sequential catalysis involving aspartate and ATP. Inhibition patterns of the four products, glutamate, AMP, PPi, and asparagine, versus each of the three substrates are consistent with a hybrid Uni Uni Bi Ter Ping Pong Theorell-Chance mechanism where the glutaminase reaction occurs first and aspartate binds to the enzyme before ATP in the sequential segment. PPi is the first product released in the Theorell-Chance reaction, which is followed by the ordered release of AMP and asparagine. Product inhibition patterns also indicate the formation of E . NH3 . Asn and E . NH3 . Asp . AMP abortive complexes. Although an amide site (for glutamine and asparagine), presumably responsible for the glutaminase reaction, an acid site (for glutamate and aspartate), and a nucleotide site are involved in the overall catalysis, the "two-site" ping-pong mechanism is incompatible with the experimentally observed product inhibition patterns.

Partial purification and properties of L-asparagine synthetase from mouse pancreas.

l-Asparagine synthetase was partially purified from mouse pancreas to a final mean specific activity of 0.10 unit/mg of protein. The enzyme exhibited an l-glutaminase activity which was not affected by l-asparate, NH(4)Cl, ATP-MgCl(2), l-glutamate, AMP (sodium salt) or sodium pyrophosphate. The l-glutamine-dependent l-asparagine synthetase activity of the partially purified enzyme from mouse pancreas was markedly decreased by freezing for 7 days at -87 degrees C in the presence of 1mm-dithiothreitol, but effectively protected from inactivation by high concentrations (10mm) of the thiol reagent. The l-glutaminase activity of the enzyme was inhibited by antagonists of l-glutamine (e.g. 6-diazo-5-oxo-l-norleucine, 5-chloro-4-oxo-l-norvaline, 5-diazo-4-oxo-l-norvaline and NSC-163501) and thiol-reactive compounds (e.g. 2-amino-4-arsenophenol hydrochloride, maleimide, mucochloric acid and ZnCl(2)), but not by aminomalonic acid, the next lower homologue of l-aspartate, nor by l-homoserine beta-adenylate, an analogue of the presumed transitory covalent intermediate. The complete forward reaction catalysed by l-asparagine synthetase from mouse pancreas appears to be irreversible and essentially stoicheiometric under the conditions examined. Mouse pancreas contains a proteolytic inhibitor of l-asparagine synthetase separable from the enzyme by ion-exchange column chromatography. The inhibitor is activated by incubation at 4 degrees C for 110h and inactivated by soya-bean trypsin inhibitor, di-isopropyl phosphorofluoridate and boiling.

Role of pancreatic L-asparagine synthetase in homeostasis of L-asparagine.

L-Asparagine synthetase from mouse pancreas was found to be associated principally with the exocrine pancreas and to be dependent on the age of the animal, but not on gender, diet, or the presence of tumor under the conditions examined. The function of the pancreatic enzyme appears to be to supply L-asparagine for the synthesis of pancreatic proteins. This function is suggested by the high specific activity of L-asparagine in pancreatic proteins after intravenous treatment of BDF1 mice with L-[U-14C]asparatate. The pancreas is also able to function as a storage depot for L-asparagine under conditions in which the concentration of the amino acid in the blood is in excess. Unlike the liver, the pancreas is unable to add L-asparagine to the circulation when the concentration of the amide is below normal limits.

Aminomalonic acid and its congeners as potential in vivo inhibitors of L-asparagine synthetase.

Aminomalonic acid is a strong in vitro inhibitor of L-asparagine synthetase from Leukemia 5178Y/AR and from mouse pancreas; the agent is formally competitive with L-aspartic acid (Ki = 0.0023 M and 0.0015 M for the tumoral and pancreatic enzymes, respectively). Since aminomalonic acid is unstable and inert in vivo as an inhibitor of L-asparagine synthetase, attempts were made to deliver it to the site of its intended action via precursors: the diamide (2-aminomalonamide), the diester (diethylaminomalonate), and the keto acid (ketomalonic acid). Each of these putative 'pro drugs' was shown to be susceptible to metabolism to aminomalonate by mammalian and bacterial enzymes, in vitro. In vivo, aminomalonamide failed to inhibit tumoral L-asparagine synthetase at any time period up to 24 h after its oral or intraperitoneal administration. The diester and keto acid were similarly inactive. However, with specialized techniques it was possible to demonstrate that the diamide significantly inhibited the amidation and/or incorporation of L-aspartic acid into the L-asparaginyl residues of protein. Chemical manipulations of aminomalonic acid aimed at introducing irreversibly reacting functions are warranted.

Potential antitumor agents via inhibitors of L-asparagine synthetase: substituted sulfonamides and sulfonyl hydrazides related to glutamine.

A series of 4-(substituted aminosulfonyl)- and 4-(substituted hydrazinosulfonyl)-2-aminobutanoic acids, compounds structurally related to glutamine, was synthesized as potential inhibitors of L-asparagine synthetase and subjected to screening as antitumor agents. Target amino acids were obtained by condensation of a blocked reactive sulfonyl chloride with the appropriate amine or hydrazide, followed by deblocking with hydrogen--palladium or liquid hydrogen fluoride--anisole. Neither the target compounds nor their protected precursors inhibited the enzyme from L5178Y/AR or prolonged the life of mice with P-388 lymphocytic leukemia. However, DL-4,4'-dithiobis[2-(benzyloxycarbonylamino)butanoic acid], an intermediate in the synthesis of the target amino acids, exhibited 90% inhibition of L-asparagine synthetase at 10 mM.

Pancreatic carcinogenesis and naturally occurring pancreatic neoplasms of rats and mice in the NCI carcinogenesis testing program.

Over two hundred chemicals were examined in a two year rodent bioassay system for possible carcinogenicity. Of these, only nitrofen significantly increased the incidence of neoplasms of the exocrine pancreas of rats or mice (female Osborne-Mendel rats); azinphosmethyl was the only agent tested which significantly increased the incidence of islet-cell tumors of rats or mice (male Osborne-Mendel rats). The use of the rat (Osborne-Mendel or Fischer 344) and mouse (B6C3F1) as models for the detection of chemically-induced pancreatic neoplasms also was investigated. The incidences of specific neoplasms of the exocrine or endocrine pancreas produced by all chemicals tested were combined and compared with the combined incidences of similar neoplasms in control animals in order to increase the sensitivity of the test. The data obtained through this procedure suggests that the male rat may be a good, sensitive model for the detection of islet-cell tumors.