Mutagenic activity of some newly synthesized cytostatic 1-(2-chloroethyl)-4-arylacyl-1-nitrososemicarbazides in the Salmonella/mammalian microsome assay.

Five experimental anti-leukemic agents, 1-(2-chloroethyl)-4-arylacyl-1-nitrososemicarbazides, were synthesized and tested for genotoxicity in the Salmonella/mammalian microsome assay. No strong mutagenic activity could be detected when tested with the S. typhimurium TA98. A clear dose-dependent base-pair-substitution mutagenic activity was observed with each compound when the tester strain TA100 was used with or without metabolic activation. The genotoxicity of the unsubstituted substance was similar to that of the known mutagenic cytostatic drugs, lomustin and carmustin, and was stronger than the mutagenicity of each substituted derivative.

Release of mutagens after chemical or microbial degradation of beech wood lignin.

The microbial or chemical degradation of lignin from untreated samples of beech wood dusts (Fagus silvatica) resulted in the release of different mutagenic responses in the Salmonella/mammalian plate incorporation assay. In the first experiment using chemical degradation of lignin, dust samples were pre-extracted using acetone-water; the lignin portions were degraded into simpler compounds which were further fractionated on a Sephadex-LH20 column. The compounds isolated from the second phase of Sephadex, representing substances with a 3-4 ring structure and/or those of the same molecular weight, were highly mutagenic towards Salmonella typhimurium TA100 in the presence of metabolic activation. These substances were also active to some extent in strain TA1537 both in the presence and absence of Aroclor-induced rat liver homogenates. In contrast, no direct- or indirect-acting mutagenicity was found when testing with strains TA97 and TA98. Strain TA1535 responded positively only to direct-acting mutagens in the fraction tested. The mutagenic fraction was found to be toxic to the cells when tested in a histidine-rich medium. Repurification of this mutagenic fraction, using silica-gel column chromatography, revealed much higher mutagenic activity than the test material towards strain TA100. In the second pilot experiment, Phanerochaete chrysosporium and Chaetomium globosum, which are known for their ability to degrade lignin, were each incubated with wood dusts in a mixture of physiological saline and nutrient broth for either 3 or 30 days. Significant mutagenic activity was observed with the dust extract after incubation with Ph. chrysosporium but not with Ch. globosum which is a known degrader of beech lignin. These results are discussed regarding hypotheses on the carcinogenicity of beech wood dusts.

Comparative carcinogenicity of cigarette mainstream and sidestream smoke condensates on the mouse skin.

The direct carcinogenic effects of sidestream (SS) and mainstream (MS) smoke condensates of a filtered commercial brand of blond cigarettes were compared using a lifetime mouse skin tumorigenicity assay on female NMRI mice. Each cigarette was smoked by a smoking machine under the standard conditions, and the separately collected SS and MS smoke condensates were extracted with acetone/methanol as described elsewhere. These were tested for carcinogenicity on an area of 1-1.5 cm shaved skin of mice on the lower back. The mice were treated with half of each dose (5, 10 or 15 mg) twice a week, for only 3 months. No substance was used as promoter or as an additional initiator of carcinogenicity. No statistically significant difference was found when the life spans of MS-treated and untreated animals were compared. In contrast, the life spans of SS-treated mice were significantly (P less than 0.01) shorter than those of MS-treated animals or those of all three negative control groups together. The observed carcinogenic effects were based on tumours and lesions found only on the site of application of the test material. Of 210 mice (effective number, 129) serving as the negative controls, 3 developed skin lesions but no tumours. Of 210 MS-treated mice (effective number, 177), 7 developed tumours (4 malignant and 3 benign) and 35 had a uniform type of precancerous skin lesions. The numbers of tumours or lesions were not increased dose-dependently. Of 210 SS-treated animals (effective number, 182), 30 developed tumours (16 malignant and 14 benign) and 56 had a uniform type of precancerous skin lesion. The initiation of these latter lesions was found to be dose-dependent (P less than 0.001). The SS-treated animals developed two to six times more skin tumours than the MS-treated mice. Comparing the negative controls with the MS- or SS-treated animals, the overall carcinogenic effect observed was statistically significant. Comparing the MS- with SS-treated animals, the overall carcinogenic effect of SS was much higher than that of MS (P less than 0.001).

Urinary and faecal excretion of pyrene and hydroxypyrene by rats after oral, intraperitoneal, intratracheal or intrapulmonary application.

The urinary and faecal excretion of pyrene and 1-hydroxypyrene after oral (53.4%), intraperitoneal (3.1%), intratracheal (30-37%) and intrapulmonary application (0.003%) to rats has been determined by means of gas chromatography/mass spectrometry and the excretion rates were found to depend on the mode of application. With regard to the low urinary excretion rates, 1-hydroxypyrene seems not to be very suitable as a biological marker for PAH exposure to man.

The mouse-skin carcinogenicity of a mutagenic fraction from beech wood dusts.

A life-time mouse-skin carcinogenicity assay was conducted using female NMRI mice to evaluate the possible direct carcinogenic activity of a mutagenic fraction isolated from beech wood dusts. The samples of untreated beech wood dusts were extracted with methanol at pH3 and were purified from the inhibitory compounds toxic to bacteria, using silica-gel column chromatography. The fraction obtained after passing through the column was tested for mutagenicity in the Ames assay employing Salmonella typhimurium TA100 in the presence of Aroclor-treated rat-liver-S9. Using acetone as the vehicle, this mutagenic fraction was tested for carcinogenicity on an area of 1-1.5 cm shaved skin of mice on the lower back. The mice were treated with half of each dose, twice a week, for only 3 months. The total doses applied per week were 2.5, 5, 7.5 or 10 g equivalent dust/mouse. No substance was used as promoter. No statistically significant difference was found when the life spans of treated and untreated animals were compared. The observed carcinogenic effect was based on tumours and lesions found only on the site of application of the test material. Of 210 mice (effective number, 129) serving as the negative controls, three developed skin lesions but no tumours. Of 280 treated animals (effective number, 188) 34 developed different types of tumours and 20 had a uniform type of precancerous skin lesion. Of 34 tumours observed 21 were originated from the skin, 12 from the mammary glands beneath the site of application, and one was a lymphoma. Comparing the negative controls with the treated animals, the overall carcinogenic effect observed was dose-dependent and statistically significant. Excluding the mammary tumours and a lymphoma found beneath the site of treatment, the overall induction of skin tumours was still significant. However, the dose-dependent increase in the number of skin tumours alone was not statistically significant. These results suggest that beech wood dust contains mutagenic and carcinogenic constituent(s).

Cancer epidemiology of woodworking.

The literature published between 1965 and 1989 on the cancer epidemiology of woodworking in furniture industries and carpentry shops in 17 countries is reviewed. Included are some unpublished data obtained through personal communication with epidemiologists or collected from doctoral dissertations. Of 5,785 cases with sino-nasal cancers, about 23% were found to be woodworkers. Dusty jobs, especially wood processing using high-speed machines, are mainly associated with the enhanced incidence of nasal adenocarcinomas. The latency periods of the latter tumors ranged from 7 to 69 years in five European countries. A variety of neoplasias of the respiratory, digestive, and urinary tracts as well as the hemopoietic and lymphatic systems, including Hodgkin's disease are reported to be significantly associated with occupational exposure to wood dust. These data suggest that the exposure to some types of wood dust might cause a systemic rather than local neoplastic disorder.

Comparative bone marrow clastogenicity of cigarette sidestream, mainstream and recombined smoke condensates in mice.

The chromosome-damaging effects of cigarette sidestream (SS) and mainstream (MS) smoke condensates and a mixture of these were compared in 8-week-old NMRI mice by intraperitoneal administration. Each filtered commercial brand of cigarette was smoked by a smoking machine under the standard conditions, and the separately collected SS and MS smoke condensates were extracted with acetone/methanol as described elsewhere. The extracts were tested before and after treatment of animals with an enzyme inducer (Aroclor 1254) or inhibitor (Metyrapone). Increased formation of micronuclei within polychromatic erythrocytes (PCEs) of femural bone marrow 30 h after injection of the extracts was regarded as being due to a clastogenic effect. Regardless of the type of smoke extract injected, the increased formation of micronuclei was found to be dose dependent. The SS smoke condensate induced approximately 29% more micronuclei than the MS smoke condensate, the difference being significant (P less than 0.01). The overall clastogenicity of a 1:1 mixture of SS and MS smoke condensates was not substantially different from the activity of either SS or MS smoke condensate alone. Pretreatment of animals with Aroclor clearly enhanced the differences between the number of micronucleated PCEs caused by SS versus MS smoke condensate; SS smoke condensate induced 50% more micronuclei than did MS smoke condensate (P less than 0.001). Pretreatment of mice with Metyrapone did not modify appreciably the induction of micronuclei by either type of smoke. These results are discussed with reference to our previous data involving inhalation experiments and the recent issue of passive smoking.

Investigations on metabolism and carcinogenicity of 1,1,2-trichloroethane.

Two groups of male and female Sprague-Dawley rats (50 animals/group per sex) were treated with either 15.37 or 46.77 mumole of 1,1,2-TCE in DMSO/rat for 2 years. The animals were treated once a week by s.c. injection of test compound in the skin of neck. Two groups of controls received either DMSO or no treatment at all. The incidence of benign mesenchymal and epithelial tumors was not significant when compared with either DMSO-treated or untreated controls. The animals treated with 46.77 mumole 1,1,2-TCE significantly developed sarcomas when compared with the untreated controls. In a further experiment, either 40 mumole or 160 mumole 1,1,2-TCE was injected into male Wistar rats and the metabolites, TdGA and HEMA, were determined in 24-h urine samples. Comparative studies were carried out giving equimolar amounts of chloroethanol and 2-chloroacetaldehyde diethyl acetal. Analysis of the metabolites showed that no detectable HEMA was excreted in urine after treatment of rats with 1,1,2-TCE or chloroethanol. TdGA was excreted in urine much more among chloroacetaldehyde-treated animals than among 1,1,2-TCE- or chloroethanol-treated rats.

Urinary and faecal excretion of chrysene and chrysene metabolites by rats after oral, intraperitoneal, intratracheal or intrapulmonary application.

The urinary and faecal excretion of chrysene and its phenolic metabolites after oral, intraperitoneal, intratracheal, and intrapulmonary administration to rats have been studied by means of gas chromatography/mass spectrometry. The metabolite profile was found to depend on the mode of excretion and on the route of administration. In all cases the oxidation of chrysene in the 1,2- or 3,4-position predominates, whereas oxidation in the 5,6-position (K-region) seems be a minor pathway.

Effects of pretreatment of male NMRI mice with enzyme inducers or inhibitors on clastogenicity of toluene.

Five groups of young male NMRI mice were pretreated with IP injections of three known inducers of cytochrome P450, Aroclor 1254, phenobarbital and 3-methylcholanthrene, and two inhibitors, metyrapone and alpha-naphthoflavone, 5, 3, 2, 1, and 1 day(s) before receiving toluene, respectively. Toluene was given to animals by IP injections of two similar doses 24 h apart. Increased formation of micronuclei within polychromatic erythrocytes of femoral bone marrow 30 h after the first injection of toluene was recorded. None of the treatments with an inducer or inhibitor alone gave a significant increase in the frequency of micronucleated polychromatic erythrocytes. However, pretreatment of animals with each inducer or even inhibitor resulted in an enhanced clastogenic activity of toluene. Simultaneous injections of an inhibitor and toluene clearly decreased the clastogenicities observed. Enhancement of the clastogenicity of toluene was more evident among Aroclor -pretreated animals than among the other groups. Treatment of animals with a mixture of toluene and benzene did not result in an additive clastogenic activity of benzene. IP injection of a mixture of toluene and every xylene isomer resulted in an enhanced clastogenic activity of toluene, although xylene isomers are not found to be clastogenic.

The bone marrow clastogenicity of eight halogenated benzenes in male NMRI mice.

Eight widely used halogenated benzenes, including bromobenzene (BB), chlorobenzene (CB), three isomers of dichlorobenzene (DCB) and three isomers of trichlorobenzene (TCB) were tested for acute toxicity (LD50) and clastogenicity in 8-week-old NMRI mice by intraperitoneal administration. Four doses of each chemical (up to 70% of LD50) were tested for clastogenic activity. Each compound was administered in two equal doses, 24 h apart. Increased formation of micronucleated polychromatic erythrocytes, observed in femoral bone marrow, 30 h after the first injection, was considered to be due to the clastogenic activity of the test compound. All the halogenated benzenes tested were found to be clastogenic (P less than 0.01). The highest clastogenic activities were induced by m-DCB and BB. Among the three isomers of DCB, m-DCB significantly (P less than 0.05) induced more micronuclei than o-DCB or p-DCB. No significant differences were found between the clastogenic activities of TCB isomers.

Urinary excretion of mutagens in smokers of cigarettes with various tar and nicotine yields, black tobacco, and cigars.

Frameshift mutagens were isolated and concentrated from smokers' urine employing a method recently described. Urine concentrates of the habitual smokers and non-smokers who smoked cigarettes with low-, medium-, and high tar/nicotine yields, RCN (Reduced Condensate and Nicotine; artificial cigarettes containing cotobacco materials), black tobacco, and cigars were tested for mutagenicity in the Salmonella/mammalian microsome assay using Salmonella typhimurium TA98. Non-smokers who smoked 5 and habitual smokers who smoked 10 cigarettes of various tar and nicotine yields excreted more mutagens in urine with low-tar cigarettes than with medium- or high-tar cigarettes. Consuming more than 10 cigarettes a day resulted in a higher urinary excretion of mutagens with medium-tar cigarettes than with high-tar cigarettes. Smoking 5 RCN cigarettes a day by habitual smokers resulted in a higher urinary excretion of mutagens than smoking 5 commercial brand of cigarettes. In contrast, smoking 10 RCN cigarettes resulted in a lower urinary excretion of mutagens than smoking 10 commercial brand of cigarettes. The highest mutagenic activity was found with the urine of a habitual black tobacco smoker. Smoking cigars by non-smokers resulted in a very weak mutagenic activity of urine.

Clastogenic effect of passive smoking on bone marrow polychromatic erythrocytes of NMRI mice.

The genotoxic effect of passive inhalation of sidestream cigarette smoke on bone marrow polychromatic erythrocytes was studied using male NMRI mice. The animals were placed in individual 145.2-dm3 glass chambers resembling a room provided with normal air flow. They were exposed to the sidestream smoke of a commercial brand of cigarettes smoked by a smoking machine under standard conditions. Increased formation of micronuclei within polychromatic erythrocytes (PCEs) of femoral bone marrow 30 h after passive smoking was regarded as being due to the clastogenic effect of the smoke. Passive inhalation of the diluted sidestream smoke of a single cigarette resulted in a significant increase (P less than 0.01) in the frequency of micronucleated PCEs. This clastogenic activity was found to be dose-dependent.

Urinary excretion of mutagens in passive smokers.

Six healthy young volunteers with no history of active smoking were asked to keep on their Western diets avoiding the consumption of alcoholic beverages, excess coffee, any sort of medicament, and the known pro- and/or anti-mutagen-containing foods and drinks, 24 h before and during the experiments. They were exposed passively to cigarette smoke produced by 4 habitual smokers in an unventilated 48.6 m3 room for 8 h. The carbon monoxide concentration was 18.85 +/- 7.3 ppm during the 8-h exposure. Frameshift mutagens were isolated from 10-h urine samples using chloroform and were tested for mutagenicity in the Salmonella/mammalian microsome assay employing Salmonella typhimurium TA98. Although clearly enhanced, no significant mutagenic activity could be found with 25 ml equivalent urine/plate after passive exposure to cigarette smoke. The weak mutagenicities found were highly significant when 50 ml equivalent urine/plate was tested. No direct correlation was observed between urine mutagenicity and the urinary cotinine concentration. The results obtained are discussed with reference to inconsistent reports in the literature concerning the mutagenicity of urine after passive smoking.

Excretion of mutagens in sweat and faeces of man, and in serum, gastric juice and urine of rats, after oral dosing of niridazole or metronidazole.

Frameshift mutagens were isolated and concentrated from sweat and faeces of three healthy volunteers (non-smokers) of whom two received 750 mg metronidazole/person and one received 500 mg niridazole. The sweat samples were collected in a sauna a day before and 8 h after oral uptake of the medicaments. The faecal samples were those expelled 12 h before and after drug uptake. All extracts were tested for mutagenicity in the bacterial microtiter fluctuation test employing Salmonella typhimurium TA1538. No mutagenic activity was found with the samples obtained before the drugs were taken, whereas the samples collected after drug treatments were all mutagenic (P less than 0.05). In an animal experiment, female Wistar rats were used to study the time-course of the excretion of mutagens in serum, urine and gastric juice after uptake of 10-20 mg niridazole by gavage. Significant mutagenic activities (P less than 0.001) were found in serum 10 min after and in gastric juice 12h after treatment with niridazole. Non-significant but detectable mutagenicities were found in urine 12 h after treatment, when S. typhimurium G46 was employed as a test organism. These latter mutagenicities were significant (P less than 0.05) 24 h after treatment, they reached a peak of activity (P less than 0.01) 48 h post-administration and disappeared 12 h thereafter.

Investigations on metabolism, genotoxic effects and carcinogenicity of 2,2'-dichlorodiethylether.

Either 40 mumole or 160 mumole 2,2'-DDE was injected into male Wistar rats and the metabolites, TdGA and HEMA, were determined in the 24-h urine specimens. Comparative investigations were carried out giving equimolar amounts of chloroethanol and 2-chloroacetaldehyde diethyl acetal. In a further step, inhalation experiments were performed to determine urinary excretion of the two metabolites after an 8-h exposure of male Wistar rats to 10, 50, 100, and 500 ppm 2,2'-DDE and to 50, 200, und 1,000 ppm vinyl chloride. A long-term study was conducted to investigate the possible carcinogenicity of 2,2'-DDE in male and female Sprague-Dawley rats following s.c. injections of 4.36 mumole and 13.1 mumole 2,2'-DDE in DMSO per week. The evaluation of tumor development in treated groups and controls were based on macroscopic inspection and histological examinations of the suspect organs and tissues. Analysis of the metabolites showed that HEMA excretion was much lower than the excretion of TdGA following the uptake of 2,2'-DDE, 2-chloroethanol and 2-chloroacetaldehyde diethyl acetal. Contrary to these, vinyl chloride uptake resulted in a higher urinary excretion of HEMA than TdGA. There was no appreciable increase in the number of tumors detected in 2,2'-DDE-treated animals when compared with untreated or DMSO-treated groups. Since irradiation of 2,2'-DDE with UV did not elevate mutagenic activity of the compound against Salmonella typhimurium TA100, the high mutagenicity of the compound found in a desiccator cannot be due to the liberation of mutagenic compounds produced under the influence of UV light.

A fraction of beech wood mutagenic in the Salmonella/mammalian microsome assay.

Base-pair substitution mutagens were isolated from the dusts of several untreated samples of beech wood and tested for mutagenicity in the Salmonella/mammalian microsome assay. These compounds reverted Salmonella typhimurium his- TA 100 in the presence of Aroclor-induced rat S9. These mutagens were found to be toxic to the cells when tested in a histidine-rich medium (complete medium). Mutagenicity of the non-fractionated wood-dust extracts due to the presence of some inhibitory compounds of wood could not be confirmed significantly. These inhibitors counteracted the reversion of bacteria when the known mutagens, such as benzo(a)pyrene, aflatoxin B1 and ethyl methanesulfonate, were tested. The results indicate that beech wood-dust contains mutagenic constituent(s) which may contribute to their assumed tumor bearing effects among wood-workers.

Effects of ethylbenzene, toluene, and xylene on the induction of micronuclei in bone marrow polychromatic erythrocytes of mice.

Genotoxic effects of five widely used aromatic industrial solvents, ethylbenzene, methylbenzene (toluene), o-, m-, and p-dimethylbenzene (xylene), on bone marrow cells of male NMRI mice were studied using micronucleus test. Each compound was given to animals by IP administration of two similar doses 24 h apart. Increased formation of micronuclei within polychromatic erythrocytes of femoral bone marrow 30 h after the first injection was conducted to be due to the clastogenic effect of the test compound. Of the chemicals tested, only toluene gave a dose-dependent increase in the frequency of micronucleated polychromatic erythrocytes. This genotoxic activity of toluene was confirmed in male B6C3F1 mice.

Determination of mutagenic activities in different fractions of automobile exhaust condensate by the Salmonella/oxygenase mutagenicity test system.

Automobile exhaust condensate of a passenger car (gasoline engine) was separated into fractions of 2-3 rings containing -, 4-7 rings containing polycyclic aromatic hydrocarbons (PAHs) and PAH-free fractions. All fractions were tested for mutagenicity by the Ames system. The highest dose-dependent increase in revertant colonies was found for the 4-7 ring PAH-fraction when tested with Salmonella typhimurium TA 98 and TA 100. These results are compatible with data obtained in in-vivo tests by previous investigations. The mutagenicity of these fractions in the absence of the oxygenase was negligible.

Isolation of frameshift mutagens from smokers' urine: experiences with three concentration methods.

Frameshift mutagens were isolated and concentrated from cigarette smokers' urine by the use of three different extraction methods; XAD-2 resin chromatography, chloroform and blue cotton extraction systems. The extracts were tested for mutagenicity in the Salmonella/mammalian microsome test employing Salmonella typhimurium TA98. By the use of XAD-2 resin chromatography, no clear correlation was found between urine mutagenicity versus number of cigarettes smoked and no detectable mutagenic activity was observed when fewer than four cigarettes a day were consumed. A clear correlation was found between urine mutagenicity versus number of cigarettes smoked when urine samples were extracted by the use of chloroform or blue cotton. Interference of free histidine in mutagenicity assays could be excluded.