Organ specific genotoxicity and carcinogenicity.

1. The in vitro studies showed that organ specific metabolic activation does not appear to play a predominant role for the in vivo activities of the studied nitrosamines 2. The in vivo studies following 1 h exposure of rats with the nitrosamines can differentiate between organs susceptible for genotoxicity and and those which are not. Nontarget organs in carcinogenicity can not be identified exclusively. 3. The additional study of persistence of genotoxicity may identify organs susceptible for carcinogenicity. Presently, we are working on new techniques to detect DNA SSB and other events with microscale methods. This is necessary to allow a more complete elucidation of genotoxicity in remote target organs and with other carcinogens which may not induce DNA SSB. Accordingly in the near future we expect to have even more versatile tools available to study toxicokinetics of foreign compound. Meanwhile our work with N-nitrosamines is continuing in order to better understand their in vivo modes of action and to better evaluate their burden and risk for man.

Employment of adult mammalian primary cells in toxicology: in vivo and in vitro genotoxic effects of environmentally significant N-nitrosodialkylamines in cells of the liver, lung, and kidney.

This report focuses on the use of freshly isolated primary mammalian cells from different tissues and organs of the rat for the rapid and efficient analysis of toxic and genotoxic chemicals. The cells are either treated in vitro or they are isolated from treated animals. Viability by trypan blue exclusion and DNA damage as single-strand breaks are monitored in either case. Therefore, it is possible to compare in vitro and in vivo results directly. N-nitrosamines with unique organ-specific modes in carcinogenesis were studied in vitro using hepatocytes derived from three species (rat, hamster, and pig) and in rat lung and kidney cells. The sensitive detection of all carcinogenic nitrosamines was achieved, although a pattern of cell-specific activation was not observable. The new modification of the in vivo approach allowed the sensitive detection of NDMA genotoxicity in hepatic and in extrahepatic tissues. It is important to point out that the method is an efficient tool for toxicokinetic studies with genotoxic carcinogens in vivo.

Assay-specific genotoxicity of N-nitrosodibenzylamine to the rat liver in vivo.

N-Nitrosodibenzylamine (NDBzA) is mutagenic to Salmonella typhimurium and induces DNA strand breaks in isolated rat hepatocytes, yet it is reported to be non-carcinogenic to the rat. Here we report that it is inactive in both the rat and mouse bone marrow micronucleus assays and in a rat liver autoradiographic assay for unscheduled DNA synthesis. It is, however, clearly active as a micronucleus-inducing agent and mitogen in the rat liver and is capable of inducing single-strand breaks in the DNA of rat liver. The origin and implications of this curious conflict of in vivo genotoxicity data are discussed. Irrespective of that discussion, it is concluded that NDBzA is genotoxic to the rat liver in vivo.

DNA amplification in genetic toxicology.

Chemical compounds can cause amplification of specific DNA sequences. DNA amplification may result in an enhanced production of gene products which help cells to cope with the chemicals. This may lead to a resistance of the cells toward the agent. Additionally, initiation of transformation or progression of transformed cells to tumorigenicity may also involve DNA amplification. Therefore, it is of interest to study the potential of chemicals to induce DNA amplification. This report focuses on the investigation of a variety of chemicals in 2 systems with which the amplification of viral DNA is measured within cells in culture. One model system comprises the measurement of SV40 DNA content in an SV40-transformed Chinese hamster cell line following chemical treatment. Antitumor agents as well as genotoxic and non-genotoxic compounds were studied in this system as a first step to determine the DNA amplification-inducing potential of a variety of differently acting chemical compounds. Also, a novel assay based on adeno-associated virus infection of cells is described. This system may offer the possibility of studying DNA amplification in a variety of different target cells. For the future, the need is stressed to develop and analyze versatile systems to study amplification of specific target genes in untransformed cells and in tumor cells.

Assessment of the preclinical activity of budotitane in three different transplantable tumor systems, its lack of mutagenicity, and first results of clinical phase I studies.

The antitumor activity of budotitane was investigated in three different tumor systems--the transplantable murine ascitic-colon-adenocarcinoma MAC 15A, the TD-osteosarcoma of the rat, and the intramuscularly transplanted murine sarcoma 180. Marked inhibition of tumor growth was observed in the intramuscularly transplanted sarcoma 180, and cure rates of 50-80% were achieved in the colon adenocarcinoma MAC 15A. In contrast to these findings, bulotitane was inactive in the transplantable TD-osteosarcoma of the rat. Preliminary mutagenicity studies with the Salmonella typhimurium/mammalian microsome assay of Ames did not show any evidence of mutagenicity for the compound. The first results of the phase I clinical trials showed mild hepatotoxicity at a dose level of 15 mg/kg, dose-limiting nephrotoxicity at 21 mg/kg, and a reversible impairment of the sense of taste, beginning at a dose of 9 mg/kg.

Effect of long-term inhalation of N-nitroso-dimethylamine (NDMA) and SO2/NOx in rats.

This report focusses on preliminary results of a long-term inhalation assay with N-nitroso-dimethylamine (NDMA) at low concentrations. Chronic inhalation of 1 ppm of NDMA (4 h/day, 5 days/week) was found to be toxic in rats and diminished life expectancy by about 8 months compared to the control group. Mostly tumors of the nasal region (25/36) were observed. Inhalation of 0.2 ppm of NDMA lead to a high tumor yield in rats (20/36). At a concentration of 0.04 ppm (= 0.12 mg/m3 in air) 3 tumors of the nasal region have been found until now. In addition, a combined inhalation study of other air pollutants SO2 or NOx together with NDMA at the 0.2 ppm level is being performed. Tumors of the nasal region have been observed in the groups with SO2 + NDMA and NOx + NDMA as well as with NDMA alone. Differences in tumor response of the groups treated with NDMA alone or in combination with SO2/NOx cannot be assessed yet. The additional treatment with the air pollutants SO2 or NOx has not affected the body weight gain or any other observable parameters of the life quality of the rats.

Anticancer activity of bisphosphonic acids in methylnitrosourea-induced mammary carcinoma of the rat--benefit of combining bisphosphonates with cytostatic agents.

This study primarily describes the cytostatic activity of a bisphosphonate and of an alkylating agent linked bisphosphonate toward mammary carcinomas in vivo. Bisphosphonates had been shown to be therapeutically active in bone metastases. There is no animal tumor model available in which both primary mammary carcinomas and bone metastases can be studied simultaneously. Therefore, the Walker carcinosarcoma model, which was used as a model for bone metastasis in earlier studies, was combined with the M-methyl-N-nitrosourea (MNU) induced mammary carcinoma as a model for the primary tumor. Four-, or six-week treatment of MNU-induced mammary carcinomas in Sprague-Dawley rats with the new aromatic bisphosphonate 4[4-[bis(2-chloroethyl)-amino]-phenyl]-1-hydroxybutane-1, 1-bisphosphonate (BAD) showed higher antitumor activity than treatment with melphalan or with 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (APD) alone. BAD is the APD moiety covalently bound to a molecule derived from melphalan. A combination therapy with 11.75 mg/kg/day APD and 0.6 mg/kg/day melphalan showed the best therapeutic efficacy in this tumor model. In comparison to monotherapy with BAD, APD, or melphalan, a significantly higher rate of complete remissions was achieved. APD, itself, was not genotoxic in 3 employed short term assays. Since bisphosphonates had been shown to be therapeutically active in bone metastases, the antitumor potency of these compounds against experimental primary mammary carcinomas, coupled with the non-genotoxicity of APD and the inhibition of osteolytic bone metastases, might be an important advancement for adjuvant chemotherapy of human mammary carcinomas.

Effects of SO2 or NOx on toxic and genotoxic properties of chemical carcinogens. I. In vitro studies.

This paper describes in vitro studies on the effects of environmental pollutants (SO2/NOx) in biological systems. Basic physical, chemical and biochemical parameters were analyzed to establish the rate of SO2/NOx absorption by the culture medium. It was shown that the pH remains constant for 24 h of exposure to gas concentrations up to 50 p.p.m. The concentration of ions resulting from absorption of each pollutant in the liquid phase is dependent on their concentration in the gas phase and on exposure time. Short exposure times and high gas dosages resulted in similar doses in the medium as long exposure periods and low gas dosages. The activities of a human serum standard (alkaline phosphatase, ALP; aspartate amino transferase, AST; alanine amino transferase, ALT; gamma-glutamyltransferase, gamma-GT; lactate dehydrogenase, LDH) were determined after gaseous exposure to SO2 and NOx. The results revealed a distinct decrease in the activity of LDH after 1, 3 and 5 h exposure to 200 p.p.m. SO2. The effects of the pollutants were assayed in vitro using fetal hamster lung cells (FHLC), rat hepatocytes and the cell line CO60. For the determination of toxic effects, it was shown that the plating efficiency was a more sensitive parameter than the assay for trypan blue exclusion. Toxicity indicated as an increase of LDH leakage was not observed from FHLC in culture. Instead, a decrease of LDH was found following SO2 exposition. This decrease was similar to that observed for the human serum standard. The induction of DNA single-strand breaks was determined as a measure of genotoxic effects. SO2 application decreased the rate of DNA single-strand breaks induced by N-nitroso-acetoxymethyl-methylamine in both FHLC and in rat hepatocytes. SO2 or NOx treatment of CO60 cells for 1 h did not result in the induction of DNA amplification. HSO3- added directly to the medium as the sodium salt, however, distinctly induced the amplification of SV40 DNA. The amplification rates induced by benzo[a]pyrene or dimethylbenzanthracene were neither influenced by SO2, NOx nor HSO3-. An additive effect of HSO3- with either benzo[a]pyrene or dimethylbenzanthracene for this biological parameter was therefore not observed.

Effects of SO2 or NOx on toxic and genotoxic properties of chemical carcinogens. II. Short term in vivo studies.

Short term in vivo studies were performed to study biological effects of the common air pollutants SO2 or NOx and their influence on the genotoxic activities of nitrosamines. Hepatocytes and lung cells were isolated from Sprague-Dawley rats which had inhaled 50 p.p.m. of SO2 or NOx for 2 weeks. After incubating the cells for 1 h, genotoxicity was determined in hepatocytes by measuring DNA single-strand breaks induced by N-nitroso-acetoxymethylmethylamine, N-nitrosodimethylamine and N-nitrosomethylbenzylamine. Parameters of toxicity (trypan blue exclusion and leakage of serum enzymes) were determined in both liver and lung cells also following 1 h incubation. The activities of aryl hydrocarbon hydroxylase (AHH), nitrosodimethylamine demethylase (NDMA-D) and glutathione-S-transferase (GST) were determined in subcellular microsomal fractions isolated from lung and liver tissues. Finally, as a measure of overall toxicity, the activities of various serum enzymes were determined in the blood serum of the rats. It was found that the induction of DNA single-strand breaks by three nitrosamines was decreased in hepatocytes from SO2-treated animals. The viability of rat hepatocytes and of rat lung cells, as determined by trypan blue exclusion, was similar in all three treatment groups immediately after isolation, as well as after 1 h incubation with DMSO or with the nitrosamines. In contrast, the leakage of enzymes was different in hepatocytes of SO2-treated rats, since lactate dehydrogenase activity was decreased. Leakage of enzymes from the lung cells did not differ from group to group, but was lower than from hepatocytes. Foreign compound metabolizing enzymes were mainly decreased in NOx-treated animals, namely AHH, NDMA-D and GST in liver and GST in the lung. For SO2-treated animals NDMA-D was increased in liver and GST was decreased in lung. Blood serum enzyme levels were not greatly different from each other, except for lactate dehydrogenase which was elevated in SO2-exposed animals.

In vivo and in vitro investigations on biological effects of aromatic bis-(2-chloroethyl)amino-bisphosphonic acids, new agents proposed for chemotherapy of bone tumors: cytostatic activity in rat osteosarcoma; toxicity and genotoxicity in liver and bone marrow; mutagenicity in S. typhimurium.

Two new aromatic bis-(2-chloroethyl)-amino derivatives (BCMP and BAD) which are linked to osteotropic bisphosphonates were investigated for their therapeutical efficacy in rat osteosarcoma. Furthermore their genotoxic potential in vitro was determined in S. typhimurium and in mammalian cells. Finally, parameters for toxicity and genotoxicity were determined in liver and bone marrow cells following in vivo treatment. It was shown that BAD was of higher therapeutic effectiveness than BCMP. Both compounds induced approximately a two fold increase of his+ revertants in S. typhimurium TA1535 following metabolic activation by subcellular liver fractions. Both compounds also induced amplification of SV40 DNA in SV40 transformed cells (CO631). This endpoint may be of importance for acquired resistancy of cells during therapy. DNA-single strand breaks were induced by BCMP but not by BAD in liver cells and CO631 cell line. Following in vivo treatment BCMP was of higher genotoxic activity in liver cells than BAD. In comparison, genotoxicity of both compounds was much lower in bone marrow cells than in liver cells. BCMP was again more potent than BAD in inducing DNA single strand breaks, whereas BAD was more toxic. The higher therapeutic efficacy of BAD together with its lower genotoxic properties makes this compound superior to BCMP as a candidate for applied chemotherapy in humans.

In vitro/in vivo effects of Mesna on the genotoxicity and toxicity of cyclophosphamide--a study aimed at clarifying the mechanism of Mesna's anticarcinogenic activity.

Cyclophosphamide is an effective antitumor agent with considerable side effects such as urotoxicity and carcinogenicity. These negative attributes may be caused by toxic and genotoxic metabolites, respectively. Mesna (sodium 2-mercaptoethane sulfonate) decreases the urotoxicity by scavenging the toxic metabolite acrolein. The present study was aimed at elucidating whether a similar scavenging of genotoxic alkylating intermediates could be found, which might cause the reduction in carcinogenicity. In vitro studies on the genotoxic and toxic properties of cyclophosphamide and its major metabolites to bacteria were therefore performed in the presence of Mesna. Mesna did not reduce the mutagenicity of any of the tested metabolites. Mesna clearly inhibited the toxic properties of acrolein. After in vivo application of Mesna and cyclophosphamide to rats, however, a lower yield of mutagens in the excreted urine was observed than after application of cyclophosphamide only.

Various short-term assays and two long-term studies with the plasticizer di(2-ethylhexyl)phthalate in the Syrian golden hamster.

The plasticizer di(2-ethylhexyl)phthalate (DEHP) and its main metabolite monethylhexylphthalate (MEHP) were investigated in several short-term in vitro assays, including mutagenicity in Salmonella typhimurium TA102, a strain sensitive to mutations arising as a cause of oxidative DNA damage. Also DNA amplification in SV40-transformed Chinese hamster cells and DNA damage in rat and hamster hepatocytes were investigated. The two compounds were not genotoxic in any of the test systems. Furthermore, DEHP was investigated in two long-term bioassays with Syrian golden hamsters using both i.p. (max. total dose 54 g/kg) and inhalative (7-10 mg/kg) application. In both experiments an additional group of animals received a combination treatment of DEHP with N-nitrosodimethylamine (NDMA). These studies were included in order to elucidate whether the observed influence of DEHP on the microsomal enzyme activity (Seth, 1982) may effect the carcinogenic activity of NDMA. There was no significant increase in tumor incidence after application of only DEHP via both routes. However, the occurrence of liver malignancies was significantly (P less than 0.001) reduced after the combination treatment in the inhalation study.

Genotoxic activities of benzamidine and its N-hydroxylated metabolite benzamidoxime in Salmonella typhimurium and mammalian cells.

The genotoxic potentials of benzamidine and benzamidoxime were determined to study the toxicological relevance of the metabolic N-oxygenation (N-hydroxylation) of benzamidines to benzamidoximes. Benzamidoxime induced DNA single-strand breaks (in rat hepatocytes) and DNA amplification in SV40-transformed hamster cells. In the experiments performed, benzamidine itself was only marginally positive in the hepatocyte/DNA single-strand break assay. Since these cells possess an intact metabolization apparatus, the biological activities may be attributed to toxic and genotoxic metabolites formed by biotransformation. In the Salmonella typhimurium mutagenicity test (TA 98 and TA 100) benzamidoxime alone exhibited a low mutagenicity in the TA 98 strain in the presence of rabbit liver S-9 fractions. These results permit recognition of the metabolic N-hydroxylation of benzamidines to benzamidoximes as a process to toxication. Indirect evidence for the formation of a glucuronide of benzamidoxime has been obtained from in vitro experiments, but it could not be established that this process was a decisive factor in the genotoxicity of benzamidoxime.

What's new in mutagenicity and carcinogenicity--status of short-term assay systems as tools in genetic toxicology and carcinogenesis.

The status of short term assay systems as tools in genetic toxicology has shifted from the goal to predict carcinogenic activity of chemicals toward their employment for understanding and elucidating the mechanisms of biological activity. This altered mode of application arises from research development in two main areas. One resulted in the observation that the calculated predictive values of individual assay systems (alone or in combination with supplementary tests) to detect carcinogens as genotoxic and non-carcinogens as non-genotoxic are lower than originally expected. The other is the increasing recognition that by employing relevant in vitro procedures, various aspects of a compound's activity can be studied which may otherwise not be clarified with available in vivo methods. This report focuses on the reasons why mutagenesis assays, or short term genotoxicity assays, in general, are not unambiguously employed for determining the carcinogenic potential of unknown compounds. One reason is that a safe prediction is not possible, since non-genotoxic carcinogens will not induce alterations of the DNA. Another reason is that even genotoxic carcinogens may not accurately respond as positive in a given test system due to the limitations of the specific assay. These are mainly seen in the incomplete metabolic conversion of the test compound, irrelevancy the measured effect may actually have for carcinogenesis, and the lack of regarding pharmacokinetic influences by a host animal, when testing in vitro. A rationale testing strategy compiled of assay systems which individually are included to overcome the restrictions is described.(ABSTRACT TRUNCATED AT 250 WORDS)

Could N-nitrosamino phosphates be transport forms of activated N-nitrosamines?

The stability of N-nitroso-N-methylamine-N-ethyl phosphate (NMEP) and N-nitroso-N-ethylamine-N-ethyl phosphate (NEEP) was studied in rat serum and in rat liver homogenate and found to be sufficiently high for the phosphates to be transported in vivo. In liver homogenates, the cleavage was more efficient, and a higher phosphatase activity was found in liver microsomal and cytosolic fractions than in serum. The substances exert a distinct activity in the Salmonella typhimurium mutagenicity test.

Analysis of N-nitrosamines for genotoxicity in primary hepatocytes derived from various species.

DNA single-strand breaks (SSB) were induced in metabolically competent primary hepatocytes from rats, hamsters and pigs by a series of N-nitrosamines with organ-specific activities. Both hepatotrophic and nonhepatotropic carcinogens were genotoxic in the liver cells of all three species. Nonhepatotropic compounds were active at relatively lower doses than liver carcinogens in all species tested, substantiating many previous findings that organ-specific activation is not the primary determining factor of organ susceptibility to cancer. In 11 experiments of almost identical quality, the degree of SSB induced by 6.25 mumol N-nitrosodimethylamine varied by 75%. This high interindividual variability, even among hepatocytes derived from a single rat strain, indicates that the slight differences seen between rat, hamster and pig hepatocytes are not necessarily due to differences in species susceptibility.

Detection of mutations in bacteria and of DNA damage and amplified DNA sequences in mammalian cells as a systematic test strategy for elucidating biological activities of chemical carcinogens.

The interdisciplinary evaluation of risks from carcinogens utilizes, inter alia, data on the activities of the compounds in short-term assays. A systematic approach is being used to determine mutagenesis in bacteria (the study of direct activities and specific modes of metabolic activation), DNA damage within primary mammalian cells (DNA single-strand breaks and persistence of damage, by a method extendable to the in vivo situation) and amplified DNA sequences in cultured cells (as an endpoint probably relevant to carcinogenesis). This test combination was expected to reduce some of the shortcomings of other batteries of tests, which suffer from a lack of appropriate metabolic conversion of compounds, irrelevancy of genetic endpoints and pharmacokinetic limitations. Furthermore, as each assay in the test strategy differs from the others only by one of the parameters described above, a reasonable understanding of divergent test results from assay to assay was anticipated. Several substances were investigated to elucidate why their activities in short-term assays and in carcinogenesis experiments do not correlate. The substances were N-nitrodimethylamine, for which formaldehyde is the reactive intermediate in bacterial mutagenesis but not in mammalian cells or in vivo, N-nitrosodiethanolamine, a carcinogen that must be activated by external alcohol dehydrogenase to be mutagenic in bacteria, N-nitrosodialkylamines, with unique organotropism in vivo for which organ-specific activation was studied in vitro, N-nitroso compounds that are inactivated in vivo but not in vitro, and components of the aristolochic acid mixture which may be metabolized oxidatively or reductively, as well as numerous miscellaneous compounds that were expected to be genotoxins on account of their chemical structure. In addition to the assessment of genotoxicity, the results obtained in individual tests of this strategy yield important data on mechanisms of activity, such as organ-specific activation and deactivation, species variations, in vitro/in vivo correlation and persistence or repair of damage.

Determination of DNA single strand breaks and selective DNA amplification by N-nitrodimethylamine and analogs, and estimation of the indicator cells' metabolic capacities.

N-nitrodimethylamine is metabolized oxidatively to N-nitrohydroxymethylmethylamine, which decomposes to yield formaldehyde and N-nitromethylamine. All four compounds and N-nitromethylamine were tested for their ability to induce DNA single strand breaks in hepatocytes and in SV 40-transformed Chinese hamster embryo cell lines. Only the two monoalkylnitramines were positive. They induced single strand breaks in hepatocytes, but were not effective in the other cells. Formaldehyde and N-nitrohydroxymethylmethylamine were toxic to the cells. None of the compounds tested was able to induce selective DNA amplification in the two transformed cell lines. Enzymes involved in drug metabolism were assayed in the hamster cell lines. The activity of UDP-glucuronosyltransferase and cytosolic epoxide hydrolase were not detectable. N-nitrodimethylamine demethylation was low. The content of reduced glutathione and the activities of glutathione transferase and membrane bound epoxide hydrolase were comparable to values obtained in the rat liver.

Use of diethyldithiocarbamate as a probe to detect stable intermediates during the decomposition of several mutagenic and nonmutagenic N-nitroso compounds.

By showing that methyldiethyldithiocarbamate is formed from the reaction of methylnitrosourea and disulfiram, we demonstrated in previous experiments that one of the anticarcinogenic/antimutagenic mechanisms of disulfiram is the scavenging of reactive species. We propose that this reaction may be employed additionally as a model for elucidating the following: (a) possible reactions between alkylating species and nucleophilic sites within the cell, and (b) the existence of stable intermediates during the metabolism of N-nitroso compounds. With structurally related pairs of nitrosoureas (n-propyl/isopropyl; cyclopropyl/allyl; 2-phenylethyl/l-phenylethyl), for which each alkylating group of the first compound can spontaneously rearrange to form the alkylating group of the second isomer, we investigated whether the alkylation proceeds via a monomolecular (sn1) or a bimolecular substitution (sn2). For this, we comparatively determined the relative mutagenic activities of each isomer in Salmonella typhimurium TA 1535, as well as their reactivities towards diethyldithiocarbamate (DDTC) by identifying the reaction products. These studies were aimed at revealing the possible formation of a free carbonium ion in the decomposition of several nitrosoureas in the rat liver supernatant fraction. Our system showed that DDTC reacts by two competing mechanisms: attack at the diazonium ion and at the free carbonium ion. Therefore the striking differences which were observed in the mutagenic potency of cyclopropylnitrosourea and N-nitrosoallylurea as well as of N-nitroso-2-phenylethylurea and N-nitroso-1-phenylethylurea cannot be explained only by the different electrophilic reactivities of the respective intermediates.