The effect of complete carcinogens on intercellular transfer of lucifer yellow in fibroblast culture

Summary The effect on permeability of gap junctions of complete powerful carcinogens, 3-methylcholanthrene (MC), 7, 12-dimethylbenz(a)anthracene (DMBA), ethyl methanesulfonate (EMS), and weak carcinogens, benz(a)anthracene (BA), benzo(e)pyrene (B(e)P) as well as the arylhydrolase inhibitor 7,8-benzoflavone (7,8-BF) has been studied with the use of a dye-coupling technique and transformed Djungarian hamster DM15 fibroblasts. MC, EMS and 7,8-BF were found to exert a strong inhibitory effect on cell-to-cell dye transfer. BA and DMBA had the uncoupling activity only in 2 out of 4 experiments. B(e)P was not shown to affect LY transfer between DM15 cells. The uncoupling effect of MC, 7,8-BF and EMS (only when EMS used at the concentration of 600 µg/ ml but not 1000 µ/ ml) appeared reversible. The causes of failure to detect DMBA and B(e)P effects on gap junctions are discussed.Abbreviations B(a)P benzo(a)pyrene - B(e)P benzo(e)pyrene - BA benz(a)anthracene - 7,8-B,F 7,8-benzoflavone - DMBA 7,12,dimethylbenz(a)anthracene - MC 3-methylcholanthrene - EMS ethyl methanesulfonate - LY Lucifer Yellow - MNNG N-methyl-N-nitro-N-nitrosoguanidine - PAH polycyclic aromatic hydrocarbons     

Combined effect of multiple carcinogens and synergy index

Tumorigenesis often involves exposure to multiple carcinogens. We here construct a two-stage model, which consists of individuals in three states:normal, intermediate, and malignant. We considered a population exposed to two carcinogens and examined two exposure scenarios: (1) two carcinogens exposed separately; and (2) carcinogens are exposed simultaneously. We calculate the incidence rate of the malignant state, introducing a "synergy index" to quantify the interaction between the two carcinogens. We confirmed the results of previous analyses, but we also calculate more general situation in which each carcinogen affects both steps.     

Induction of hyperplasia and anaplasia by carcinogens in organ cultures of mouse prostate

Summary In an effort to establish a test system to examine the carcinogenic potential of chemicals, mouse prostate explants were maintained as organ cultures and the effects of carcinogenic and noncarcinogenic compounds were examined at various intervals after treatment. The degree of hyperplasia produced by a compound was determined by the colcemid metaphase arrest technique. Extensive hyperplasia of the prostatic epithelium occurred at 8 days after treatment with 3-methylcholanthrene, the 11–12 epoxide of methylcholanthrene, benzo(a)pyrene and N-methyl-N-nitro-N-nitrosoguanidine. At 12 days most carcinogentreated explants were anaplastic. The noncarcinogenic compounds, pyrene and phenanthrene, did not produce a mitotic stimulatory effect on the epithelium of the explants. The data suggest that the organ culture system of mouse prostate may be employed as a test system to obtain preliminary information regarding the carcinogenicity of a compound. This investigation was supported by Contract No. NO1-CP-22064 from the Lung Cancer Segment, Division of Cancer Cause and Prevention, National Cancer Institute, NIH, Department of Health, Education and Welfare.     

Quantitative evaluation of the effects of human carcinogens and related chemicals on human foreskin fibroblasts

Ten compounds representative of diverse classes of chemicals were evaluated for their cytotoxicity and transforming ability to human skin fibroblasts in vitro. Only five of the ten compounds were highly cytotoxic in the 0-100 µg/ml range and their order of cytotoxicity was: 2,5-bis(1-aziridinyl)-3,6-bis(carboethoxyamino)-1,4-benzoquinone (AZQ) > cis platin > bis(chloromethyl)ether (BCME) > acrylonitrile > afatoxin BI (AFBI). The other five compounds, afatoxin B2 (AFB2), methylmethacrylate, 1-naphthylamine (1-NA), 2-naphthylamine (2-NA), and cyclophosphamide, exhibited less than 40% inhibition of colony formation even at 100 µg/ml of the compound (the maximum concentration of AFB2 used was 50 µg/ml due to its low solubility). Anchorage-independent growth of exposed cells in soft agar was used as a biological endpoint for the expression of chemical transformation. AFB₁ had strong transforming ability, whereas AFB₂ was a weak transforming agent. The transforming abilities of acrylonitrile, AZQ, BCME, cis-platin, methylmethacrylate and 2-NA ranged between those of AFBI and AFB2. 1-NA also induced the soft agar growth property in the treated cells even though this compound has not been shown to be carcinogenic. AFB₁, AZQ, cisplatin, cyclophosphamide and 1-NA exhibited a dose dependent increase in soft agar growth frequency for at least three consecutive concentrations. The data suggest that anchorage-independent colony forming ability of exposed cells is a reliable marker to measure the carcinogenic potential of various hazardous chemicals.Abbreviations AZQ 2,5-bis(1-aziridinyl)-3,6-bis(carboethoxyamino)-1,4-benzoquinone - AFB aflatoxin B₁ - AFB2 aflatoxin 132 - AI anchorage independent - B[a]P benzo[a]pyrene - BCME bis(chloromethyl)ether; cis-platin, cis-diammine-dichloroplatinum - CM complete medium - E.D.50 effective dose which produced 50% cytotoxicity - CP cyclophosphamide - HNF human neonatal foreskin - HPLC high pressure liquid chromatography - 1-NA 1-naphthylamine - 2-NA 2-naphthylamine - PDL population doubling     

The unique role of rodents in the detection of possible human carcinogens and mutagens

Some of the probable reasons underlying the observation that not all chemicals shown to be genotoxic in vitro are capable of eliciting tumours in rodents or humans are discussed using appropriate examples. It is suggested that a substantial proportion of the resources currently available for conducting rodent carcinogenicity bioassays should be employed in the short-term evaluation in vivo of some of the many hundreds of chemicals recently defined as genotoxic in vitro, rather than in the protracted evaluation of a few chemicals, often of unknown activity in vitro, for carcinogenicity. A decision tree approach to the evaluation of chemicals for human mutagenic/carcinogenic potential is presented which is at variance with the construction and philosophy of many of the current legislative guidelines. The immediate need for the adoption of one of the available short-term in vivo liver assays, and/or the development of a short-term in vivo rodent assay capable of concomitantly monitoring different genetic end-points in a range of organs or tissues is emphasized.     

Effect of cigarette smoke on the mutagenic activation of environmental carcinogens by rodent liver.

In order to assess the effect of cigarette smoke (CS) on metabolic enzymes, male hamsters and rats were exposed for two weeks to smoke produced in a Hamburg type II smoking machine. The livers were then used for Ames liquid incubation and western immunoblot assays. Mutagenic activities of seven heterocyclic amines (HCAs) in Salmonella typhimurium TA98 in the presence of rat or hamster liver S9 were elevated up to 3.7 times above controls (including sham smoke control). Enhancement of mutagenic activities of PhIP and aflatoxin B(1) was observed only in CS-exposed hamster, whereas no significant alteration of mutagenicity was observed with 2-aminofluorene, benzo[a]pyrene, and 3'-hydroxymethyl-N, N-dimethyl-4-aminoazobenzene in strain TA98 or with six N-nitrosodialkylamines in strain TA100. 7,8-Benzoflavone and/or furafylline considerably inhibited the mutagenic activation of IQ and Trp-P-1 in the presence of liver S9 from untreated hamsters and sham smoke- or CS-exposed hamsters and rats, indicating the predominant involvement of hamster cytochrome P450 (CYP) 1A enzymes in the metabolic activation of HCAs. In addition, the data suggest that CS-exposure may selectively induce hepatic CYP1A1/1A2 isoforms. Western immunoblot analyses of liver microsomes using anti-rat CYP antibodies revealed that CS-exposure increased the levels of hamster CYP1A2 (3.9-fold) and rat CYP1A2 (3.0-fold) and CYP1A1, without significant change in the levels of CYP2E1 and CYP2B and 3A isoforms in each species. The presently observed selective induction of HCA activation and CYP isozymes due to CS supports the idea that CS may contribute to enhancing effects on initiation by carcinogens which are metabolically activated by hepatic CYP1A1/1A2. In conjunction with results observed for smokers, the present findings indicate that the hamster is a good animal for studies with CS, and that cigarette smoking in combination with intake of heating protein-rich foods as a life style may markedly contribute to the human carcinogenesis by HCAs.     

Identification of potential bladder progenitor cells in the trigone

Urothelial cells are specialized epithelial cells in the bladder that serve as a barrier toward excreted urine. The urothelium consists of superficial cells (most differentiated cells), intermediate cells, and basal cells; the latter have been considered as urothelium progenitor cells. In this study, BrdU or EdU was administrated to pregnant mice during E8–E13 for 2 consecutive days when bladder development occurs. The presence of label retaining cells was investigated in bladders from offspring. In 6 months old mice ~1% of bladder cells retained labeling. Stem cell markers as defined for other tissues (e.g., p63, CD44, CD117, trop2) co-localized or were in close vicinity to label retaining cells, but they were not uniquely limited to these cells. Remarkably, label retaining cells were distributed in all three cell layers (p63+, CK7+, and CK20+) of the urothelium and concentrated in the bladder trigone. This study demonstrates that bladder progenitor cells are present in all cell layers and reside mostly in the trigone. Understanding the geographic location of slow cycling cells provides crucial information for tissue regenerative purposes in the future.     

Mutagenicity of the human bladder carcinogen 4-aminobiphenyl to the bladder of Muta ™Mouse transgenic mice

The human carcinogen 4-aminobiphenyl (4AB) was evaluated in the Muta ™Mouse transgenic mouse mutation assay. A single oral dose of 75 mg/kg induced 6.9-, 1.8- and 2.2-fold increases in the mutation frequency (MF) in the bladder, liver and bone marrow, respectively. Ten daily oral doses of 10 mg/kg 4AB increased the MF in the bladder, liver and bone marrow by 13.7-, 4.8- 2.4-fold the control value, respectively. The repeat dosing protocol was therefore more sensitive than the single dose protocol. Assessment of DNA samples prepared from pooled liver homogenates clearly indicated the increase in MF observed in the individual liver samples obtained from both groups of 4AB-treated mice.     

The in vitro interaction of the carcinogens, 4-nitroquinoline-N-oxide and its metabolite 4-hydroxylaminoquinoline-N-oxide, with intact rat liver mitochondria.

The inhibition of rat liver mitochondrial respiration caused by rotenone, is relieved by the 2 carcinogens, 4-nitroquinoline-N-oxide (NQO) and its metabolite 4-hydroxylaminoquinoline-N-oxide (HAQO). Thus these agents cause reducing equivalents to circumvent the first coupling site of the respiratory chain. This is another example of the experimental confluence between oxidative phosphorylation and chemical carcinogenesis.     

Endorphins and the central inhibition of urinary bladder motility

The involvement of endogenous opioid mechanisms in the central neurogenic control of urinary bladder function has been examined in anesthetized rats. Intracerebroventricular (ICV) microinjection of β-endorphin (0.5–2.0 μg) produced powerful inhibition of rhythmic bladder contractions initiated by central reflex activity. The peptide fragments γ-endorphin and α-endorphin (4–16 μg), formed by the processing of β-endorphin by membrane homogenates of brain, were less active than the parent compound. The inhibitory effects of β-endorphin was reversed by ICV naloxone (1–2 μg) but higher doses were required to reverse γ- or α-endorphin effects. ICV naloxone administered alone increased intravesicular pressure and bladder contraction frequency. These observations support the hypothesis that the endorphins have a physiological role in the central regulation of urinary bladder activity.     

The use of biomarkers in the antioxidant responses of Daphnia magna to the acute and chronic exposure to no. 20 diesel oil and 2,4-dichlorophenol

Abstract

The effects of no. 20 diesel oil exposure, 2,4-dichlorophenol (2,4-DCP) exposure and combined exposure on the antioxidant defences of Daphnia magna have been studied systematically for the first time. Daphnia magna was exposed for 1 day or 10 days to several concentrations of 0, 0.005, 0.01, 0.05, 0.1, 0.5 or 1.0 mg L^?1 solutions. Antioxidant defences consisting of the levels of reduced glutathione (GSH) and glutathione disulfide (GSSG) and activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), selenium dependent glutathione peroxidase (Se-GPx) and glutathione S-transferase (GST) of daphnids were determined to evaluate their protective roles and to analyse the occurrence of oxidative stress. The possible antioxidant defence mechanisms are discussed. Furthermore, GST can be a potential biomarker and an early-warning index for the pollutants in waters in that GST responded sensitively to 1 day and 10 days of exposure to diesel oil and 2,4-DCP and 10 days of combined exposure. Crossover comparisons showed an antagonistic action about the no-observed-effect concentration (NOEC) against Daphnia magna, which needs further studies.     

The use of tumors in wild populations of fish to assess ecosystem health

Evidence has linked toxicants in aquatic systems with cancer in fish and population level effects on species. Thus some types of tumors may be useful monitors of ecosystem health, at least as affected by genotoxins and promoters. However, tumors caused by purely genetic mechanisms or by virus would not be good indicators. Only neoplasms which have chemicals as a portion of their etiology (either as initiators or promoters) would be useful in assessing ecosystem health. Lesions which may fit these criteria include liver neoplasms (both biliary and hepatic) and skin lesions in a variety of primarily benthic fishes, and neural lesions in various drum species and in butterfly fish species. Two studies purporting to demonstrate a lack of tumors in fish from polluted areas have been reexamined and found either to have insufficient data on vulnerable species or to actually support a tumor-pollution linkage. Thus certain lesions in vulnerable species or species groups may serve as a mechanism to assess one facet of ecosystem health.     

The resistance of epithelia to vascularization: Proteinase and endothelial cell growth inhibitory activities in urinary bladder epithelium

The avascularity of epithelia may be attributed to the presence of an extractable, low-molecular-weight factor. This factor contains potent inhibitors of proteolytic enzymes, as well as a growth inhibitory activity directed against endothelial cells in vitro. It is extracted from the epithelium of bovine urinary bladders by 1 M NaCl. The extract is ultrafiltered through an Amicon XM-50 membrane, then concentrated and dialyzed into a 0.9% NaCl solution, using a UM-2 membrane. This ultrafiltrate, called the UM-2 retentate (UM-2R). contains approximately 6 μg protein/ g tissue. The UM-2R has a low content of uronic acid and is practically devoid of hydroxyproline. SDS-PAGE reveals that the UM-2R consists of six major proteins. The UM-2R contains a Trasylol-like proteinase inhibitor that expresses strong trypsin inhibitory activity. Comparisons between bladder and serum UM-2Rs and electrophoretic mobility assays indicate that this proteinase inhibitory activity is derived from the bladder epithelium and not from the serum. The UM-2R is cytotoxic to cultured endothelial cells. Cultures of other cell types (normal and neoplastic) are not affected. The bladder-derived proteinase and endothelial cell growth inhibitory activities may protect epithelia from vascular invasion.     

Human cytochrome-P450 enzymes metabolize N-(2-methoxyphenyl)hydroxylamine, a metabolite of the carcinogens o-anisidine and o-nitroanisole, thereby dictating its genotoxicity

N-(2-Methoxyphenyl)hydroxylamine is a component in the human metabolism of two industrial and environmental pollutants and bladder carcinogens, viz. 2-methoxyaniline (o-anisidine) and 2-methoxynitrobenzene (o-nitroanisole), and it is responsible for their genotoxicity. Besides its capability to form three deoxyguanosine adducts in DNA, N-(2-methoxyphenyl)-hydroxylamine is also further metabolized by hepatic microsomal enzymes. To investigate its metabolism by human hepatic microsomes and to identify the major microsomal enzymes involved in this process are the aims of this study. N-(2-Methoxyphenyl)hydroxylamine is metabolized by human hepatic microsomes predominantly to o-anisidine, one of the parent carcinogens from which N-(2-methoxyphenyl)hydroxylamine is formed, while o-aminophenol and two N-(2-methoxyphenyl)hydroxylamine metabolites, whose exact structures have not been identified as yet, are minor products. Selective inhibitors of microsomal CYPs, NADPH:CYP reductase and NADH:cytochrome-b(5) reductase were used to characterize human liver microsomal enzymes reducing N-(2-methoxyphenyl)hydroxylamine to o-anisidine. Based on these studies, we attribute the main activity for this metabolic step in human liver to CYP3A4, 2E1 and 2C (more than 90%). The enzymes CYP2D6 and 2A6 also partake in this N-(2-methoxyphenyl)hydroxylamine metabolism in human liver, but only to ～6%. Among the human recombinant CYP enzymes tested in this study, human CYP2E1, followed by CYP3A4, 1A2, 2B6 and 2D6, were the most efficient enzymes metabolizing N-(2-methoxyphenyl)hydroxylamine to o-anisidine. The results found in this study indicate that genotoxicity of N-(2-methoxyphenyl)hydroxylamine is dictated by its spontaneous decomposition to nitrenium/carbenium ions generating DNA adducts, and by its susceptibility to metabolism by CYP enzymes.     

Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens III. Appropriate follow-up testing in vivo

There has been much discussion in recent years regarding the most appropriate follow-up testing in vivo when positive results are obtained in vitro but the in vivo micronucleus (MN) test (traditionally the most widely-used test) is negative. Not all rodent carcinogens give positive results in the micronucleus test, and so it has been common practice to include a second in vivo assay such as the unscheduled DNA synthesis (UDS) test. This has proved useful but is usually limited to analysis of rodent (usually rat) liver. With the increased evaluation and use of other in vivo assays, e.g. for transgenic mutations (TG) and DNA damage (Comet assay) it was important to investigate their usefulness. We therefore examined the published in vivo UDS, TG and Comet-assay results for 67 carcinogens that were negative or equivocal in the micronucleus test. Between 30 and 41 chemicals were evaluated in each of the three in vivo tests, with some overlap. In general, the UDS test was disappointing and gave positive results with <20% of these carcinogens, some of which induced tumours in rat liver and produced DNA adducts in vivo. The TG assay gave positive responses with >50% of the carcinogens, but the Comet assay detected almost 90% of the micronucleus-negative or equivocal carcinogens. This pattern of results was virtually unchanged when the in vitro profile (gene mutagen or clastogen) was taken into account. High sensitivity (ability to detect carcinogens as positive) is only really useful when the specificity (ability to give negative results with non-carcinogens) is also high. Based on small numbers of publications with non-carcinogens, the TG and Comet assays gave negative results with non-carcinogens on 69 and 78% of occasions, respectively. Although further evaluation of the Comet and TG assays, particularly with non-carcinogens, is needed, these data suggest that they both should play a more prominent role in regulatory testing strategies than the UDS test.