The scientific and methodological bases of experimental studies for detecting and quantifying carcinogenic risks.

This paper outlines the aims and potential scope of experimental research for risk identification and assessment in industrial carcinogenesis (environmental and occupational). It then reviews the basic, general, and specific requisites of a rigorously scientific nature that are required to render experiments to be more appropriate and better geared to the information they seek. A range of experimental approaches to risk assessment are illustrated by results achieved in the Cancer Research Centre of the Ramazzini Foundation (CRC/RF). The paper ends with a call for closer relations and integration among experimental, epidemiologic, and biostatistical studies.

Mega-experiments to identify and assess diffuse carcinogenic risks.

Diffuse carcinogenic risks, that is, those of low potency involving large areas of population and sometimes all mankind, pose a serious public health problem. Controlling these risks might help to reduce the incidence of, and mortality from, cancer. Because of their low expected carcinogenic potential, these risks are difficult to expose or assess. Epidemiologic investigation is of limited use in this field and yields its data too late to be useful. Experimental studies offer the only possible approach for assessing such risks. To increase experimental sensitivity and consistency of results, mega-experiments must be designed. That is, experiments that use a large number of animals with a well-known basic tumorigram, that extend the exposure and the biophase for as long as possible, that carefully observe the effects, and that are performed with suitable standardized methods. In the last 15 years the Ramazzini Foundation, in its Cancer Research Center at Bentivoglio, has conducted or planned five mega-experiments. Initial results indicate the great potential of these methods for identifying and assessing diffuse risks.

Prevention of breast cancer with tamoxifen: preliminary findings from the Italian randomised trial among hysterectomised women. Italian Tamoxifen Prevention Study.

BACKGROUND: Tamoxifen is a candidate chemopreventive agent in breast cancer, although the drug may be associated with the development of endometrial cancer. Therefore we did a trial in hysterectomised women of tamoxifen as a chemopreventive. METHODS: In October, 1992, we started a double-blind placebo-controlled, randomised trial of tamoxifen in women (mainly in Italy) who did not have breast cancer and who had had a hysterectomy. Women were randomised to receive tamoxifen 20 mg per day or placebo, both orally for 5 years. The original plan was to follow the intervention phase by 5 years' follow-up. In June, 1997, the trialists and the data-monitoring committee decided to end recruitment primarily because of the number of women dropping out of the study. Recruitment ended on July 11, 1997, and the study will continue as planned. The primary endpoints are the occurrence of and deaths from breast cancer. This preliminary interim analysis is based on intention-to-treat. FINDINGS: 5408 women were randomised; participating women have a median follow-up of 46 months for major endpoints. 41 cases of breast cancer occurred so far; there have been no deaths from breast cancer. There is no difference in breast-cancer frequency between the placebo (22 cases) and tamoxifen (19) arms. There is a statistically significant reduction of breast cancer among women receiving tamoxifen who also used hormone-replacement therapy during the trial: among 390 women on such therapy and allocated to placebo, we found eight cases of breast cancer compared with one case among 362 women allocated to tamoxifen. Compared with the placebo group, there was a significantly increased risk of vascular events and hypertriglyceridaemia among women on tamoxifen. INTERPRETATION: Although this preliminary analysis has low power, in this cohort of women at low-to-normal risk of breast cancer, the postulated protective effects of tamoxifen are not yet apparent. Women using hormone-replacement therapy appear to have benefited from use of tamoxifen. There were no deaths from breast cancer recorded in women in the study. It is essential to continue follow-up to quantify the long-term risks and benefits of tamoxifen therapy.

Mesotheliomas in some selected Italian population groups.

The analysis of 335 cases of mesothelioma observed at the Ramazzini Foundation and the Bologna Institute of Oncology has shown: 1) a high percentage of correlation of these tumours with asbestos exposure; 2) a large number of population categories potentially exposed to asbestos fibres and therefore at risk of developing mesothelioma; and 3) the high risk of mesothelioma among people exposed in various circumstances to asbestos used in railroads and sugar refinery plants.

p53 gene mutation pattern in rat liver tumors induced by vinyl chloride.

Vinyl chloride (VC) induces angiosarcomas of the liver (ASL) and hepatocellular carcinomas (HCCs) in humans and rodents. We examined the presence of p53 gene mutations in ASL and HCC induced by VC in Sprague Dawley rats; 25 ASL and eight HCCs were analyzed for point mutations in exons 5-8, using PCR amplification, single-strand conformation polymorphism analysis, and direct DNA sequencing. Mutations were found in 11 (44%) of the ASL and in 1 HCC. A 12-base pair deletion was found in one tumor; all others were base pair substitutions. Nine of the point mutations were observed at A:T base pairs (5 A:T --> T:A; 2 A:T --> G:C, and 2 A:T --> C:G), and of three G:C --> A:T transitions, only one was at a CpG site. In ASL, four mutations were found in exon 5, two in exon 6, and six in exon 7; the base pair substitution found in one HCC was in exon 8. One ASL exhibited two point mutations, including a silent one. Two ASL exhibited the same mutation in codon 203 and two other samples in codon 253. Codon 235 was found to be mutated in three ASL. These data show that p53 is often mutated in ASL induced by VC in rats and, as observed in ASL in humans exposed to VC, the majority of the missense mutations involved A:T base pairs. The characteristic patterns of mutations found suggest that a common mechanism operates in VC-induced p53 mutagenesis in both species, and these mutations are consistent with the formation of DNA etheno adducts by VC in the liver. The A:T --> T:A transversion observed in the first nucleotide of codon 253 in two rat ASL is equivalent to the A:T --> T:A transversion characterized previously in codon 255 in one human ASL associated with VC exposure.

Chemoprevention trial of contralateral breast cancer with fenretinide. Rationale, design, methodology, organization, data management, statistics and accrual.

The Fenretinide (4-HPR) Breast Cancer Study is a randomized multicenter clinical trial originally designed and conducted by the investigators of the Istituto Nazionale Tumori of Milan. The study is sponsored by the National Cancer Institute of Bethesda and by the Italian National Research Council. The trial was designed to evaluate the effectiveness of the synthetic retinoid 4-HPR, at a dose of 200 mg per os every day for 5 years, in reducing the incidence of contralateral breast cancer in a population of patients previously operated on for breast cancer. Between 1987 and 1993, the Istituto Nazionale Tumori of Milan and 9 other collaborating Centers enrolled 2,972 women between the ages of 30 and 70 years who had been previously operated on for T1-T2 N- M0 breast cancer. This paper describes the rationale, design, methodology, organization, data management, statistics and accrual of the participating population.

Results of long-term experimental carcinogenicity studies of the effects of gasoline, correlated fuels, and major gasoline aromatics on rats.

Unleaded gasoline, with high aromatic content, leaded gasoline, gasoil (diesel), kerosene, toluene, xylenes, ethylbenzene, and 1,2,4-trimethyl-benzene were submitted to long-term experimental carcinogenicity bioassays. The mixtures and the compounds were administered by stomach tube, in olive oil, once daily, 4 days weekly, for 104 weeks, to male and female Sprague-Dawley rats. The animals were kept under control until the end of the experiments. With varying degrees of evidence, all the tested materials were found to increase the total number of malignant tumors and of some site-specific tumors. They must therefore be considered carcinogenic. On the basis of our results the rank of carcinogenic potency of the tested aromatic hydrocarbons increases in the following order: 1,2,4-trimethylbenzene, ethylbenzene, xylenes, toluene (benzene).

Results of long-term experimental studies on the carcinogenicity of methyl tert-butyl ether.

Methyl-tert-butyl ether (MTBE) was submitted to long-term carcinogenicity bioassays on Sprague-Dawley rats. The test compound was delivered in olive oil by stomach tube (gavage), at the doses of 1000, 250, and 0 mg/kg b.w. to groups of 60 males and 60 females, once daily, 4 times weekly, for 104 weeks. All animals were kept under control until spontaneous death. MTBE was found to cause in males an increased incidence of Leydig cell testicular tumors in the group treated with the higher dose, and in females a dose-related increase of leukemias, an increase of dysplastic proliferations of lymphoreticular tissues, and also an increase of uterine sarcomas at the lower tested dose. On the basis of the presented data, MTBE must be considered a potential carcinogen.

Results of long-term carcinogenicity studies of chlorine in rats.

Four groups, each of 50 male and 50 female Sprague-Dawley rats, of the colony used in the Cancer Research Center of Bentivoglio of the Ramazzini Foundation, 12 weeks old at the start of the study, received drinking water containing sodium hypochlorite, resulting in concentrations of active chlorine of 750, 500, and 100 mg/l (treated groups), and tap water (active chlorine < 0.2 mg/l) (control group), respectively, for 104 weeks. Among the female rats of the treated groups, an increased incidence of lymphomas and leukemias has been observed, although this is not clearly dose related. Moreover, sporadic cases of some tumors, the occurrence of which is extremely unusual among the untreated rats of the colony used (historical controls), were detected in chlorine-exposed animals. The results of this study confirm the results of the experiment of the United States National Toxicology Program (1991), which showed an increase of leukemia among female Fischer 344/N rats following the administration of chlorine (in the form of sodium hypochlorite and chloramine) in their drinking water. The data here presented call for further research aimed at quantifying the oncogenic risks related to the chlorination of drinking water, to be used as a basis for consequent public health measures.

Results of a long-term experimental study on the carcinogenicity of vinyl acetate monomer in mice.

Vinyl acetate monomer (VAM) was administered in drinking water at doses of 5,000, 1,000, and 0 ppm (v/v), to Swiss mice, 17 weeks old (breeders) or 12-day embryos (offspring) at the start of the experiment. The treatment lasted 78 weeks, and the animals were kept under control until spontaneous death. VAM has been shown to cause an increase in: (1) total malignant tumors; (2) carcinomas of the Zymbal glands, oral cavity, tongue, esophagus, and forestomach; (3) stomach tumors; (4) lung tumors; and (5) uterine tumors. A slight increase of hepatomas has been observed among male mice offspring treated with the higher dose. On the basis of these data VAM must be considered a multipotential carcinogen.

Results of three life-span experimental carcinogenicity and anticarcinogenicity studies on tamoxifen in rats.

Tamoxifen was submitted to carcinogenicity bioassays on Sprague-Dawley rats (of the colony used at the Cancer Research Center in the Castle of Bentivoglio of the European Ramazzini Foundation of Oncology and Environmental Sciences) at the dose of 3.3 mg/kg b.w., by stomach tube, in three experiments. In the first experiment the drug was administered once daily, 6 days a week to male and female rats, 8 weeks old at start for their life span. In the second experiment, the drug was administered to female rats, 12 weeks old at start, once daily for 8 consecutive days every 8 weeks for their life span. In the third experiment the drug was administered to female rats, 56 weeks old at start, 6 times weekly for 40 weeks; and then the animals were kept alive for their life span. In the first experiment, a mild increase in hepatocarcinomas with low grading was detected. In the first and second experiments, a borderline increase in uterine malignancies was found. No carcinogenic effect was observed in the third experiment. In the three experiments, tamoxifen showed a strong, long-lasting chemopreventive effect on mammary benign tumors and cancers. The presented data also indicate that tamoxifen treatment reduces the incidence of other tumors: pituitary adenomas, adrenal pheochromocytomas, islet cell pancreatic tumors, Leydig cell testicular tumors, and polyps of the uterus.

Mesotheliomas following exposure to asbestos used in railroads: 130 Italian cases.

The available knowledge on the oncogenic risks of asbestos, the data on the uses of asbestos in railroads, with particular regard to the Italian State Railroads (Ferrovie dello Stato = FS), and the groups at risk due to the exposure to asbestos used in railroads were briefly reviewed. The available data on the pathological effects of such exposure, and particularly on the onset of mesotheliomas among machinists and other railroad workers, were also summarized. One hundred and thirty cases of mesothelioma (122 pleural, 1 pericardial, 6 peritoneal and 1 pleuro-peritoneal), related to the exposure to asbestos used in railroads, observed in various Italian regions, were reported. Fifty-three of these cases (among which 49 reported in the Emilia Romagna Region) were submitted to a detailed study at the Bologna Institute of Oncology. Seventy-seven cases of mesothelioma occurred among occupationally exposed FS workers, in particular machinists; 45 cases occurred among rolling-stock machinists and workers engaged in the repair and demolition of the rails of workshops not belonging to the FS; 3 cases occurred among travelling workers of rolling-stock, not belonging to the FS; and 5 cases were found in family members (1 daughter, 3 wives and 1 sister) of railroad workers. This series of cases, together with similar data from the literature, proves the existence of an actual health risk due to asbestos used in railroads, and indicates its gravity. On the basis of the available data, the following steps are considered necessary: the promotion of systematic epidemiological investigations, the adoption of preventive measures, the performance of medical oncological surveillance, and the automatic compensation for tumours following the exposure to the asbestos used in railroads.

Mesotheliomas following exposure to asbestos used in sugar refineries: report of 12 Italian cases.

Twelve Italian cases of mesothelioma (all the cases but one from the Emilia Romagna Region), following exposure to asbestos used in sugar refinery plants, are reported. Eleven cases arose in workers occupationally exposed, and one in the daughter of an exposed worker, following family contact. Eleven of the cases were pleural and one peritoneal. In the 11 cases following occupational exposure the average latency time was 36.0 (range 23-48) years, and the average age at onset was 63.4 years. In the case which followed family contact, the latency time and the age of the onset were 37 years. This represents the largest series of cases of mesothelioma due to the asbestos present in sugar refinery plants reported to date in the scientific literature. While these cases demonstrate the risk of asbestos mesothelioma in the sugar refinery industry, they in no way give the dimension of the pathological effects of asbestos (and man-made mineral fibres) used in this industry. To assess this risk further research is suggested.

Ignored occupational risks of asbestos mesotheliomas.

Nine cases of asbestos mesothelioma following usually ignored asbestos exposure were reported. In these cases the asbestos exposure has been traced following a thorough medical inquiry within the hospital. Such a type of inquiry would reduce the number of mesotheliomas due to unknown causes or considered spontaneous. The role of the hospitals as a primary source of epidemiological information is stressed.