Giuseppe Della Porta

Sister-chromatid exchanges in lymphocytes and mutagenicity in urine of nurses handling cytostatic drugs

Sister-chromatid exchanges (SCE) in peripheral blood lymphocytes and mutagenicity of urine (Ames test) were measured in a group of 21 nurses professionally handling antineoplastic drugs and in a group of 21 unexposed controls. No differences in SCE frequencies and in urinary mutagenic activity between exposed and unexposed groups were detected. A clear positive increase in urinary mutagenicity in the TA98 Salmonella strain was observed with increasing number of cigarettes smoked, whereas no evident influence of smoking on SCE was seen. Age, coffee and alcohol consumption did not show any detectable effect in the two tests.

Genetic susceptibility to murine hepatocarcinogenesis is associated with high growth rate of NDEA-initiated hepatocytes

SummaryThe murine hybrids (C57BL/6JxC3Hf)F1 (B6C3) and (C57BL/6JxBALB/c)F1 (B6C), which have a high and low spontaneous and induced incidence of hepatocellular tumors, respectively, were treated with a single dose of NDEA at 1 week of age followed by TCPOBOP, a phenobarbital-like promoter of liver carcinogenesis, or by vehicle, and sacrificed at 30 weeks of age. The frequency per liver of hepatocellular nodules was similar in the two hybrids. However, in male mice the mean volume of nodules was about 10-fold greater in B6C3 than in B6C mice receiving NDEA followed by vehicle, and the treatment with TCPOBOP after NDEA stimulated nodule growth, with a much greater response in B6C3 mice. In female mice no differences in the mean volume of nodules were seen between hybrids after NDEA and vehicle, whereas upon NDEA and TCPOBOP treatment the mean volume of nodules was 25-fold greater in B6C3 than in B6C females. In addition, a few hepatocellular adenomas and carcinomas were observed, mostly in animals treated with NDEA and TCPOBOP, and they were 3-fold more numerous among B6C3 than B6C mice. TCPOBOP alone induced the same biochemical and hyperplastic effects in the liver of both hybrids. Using DNA probes homologous to Moloney murine leukemia virus, intracisternal A particle and virus-like 30S sequences, no correlation was apparent between the expression of any of these endogenous retroviral families and the strain susceptibility to hepatocarcinogenesis. We hypothesize that the different susceptibility to hepatocarcinogenesis between B6C3 and B6C mice is related to a higher growth rate of B6C3 than B6C initiated liver cells.

Enhancing effects of carbon tetrachloride in mouse hepatocarcinogenesis

The enhancing effects of 4 or 8 necrogenic doses of carbon tetrachloride (CCl4) on hepatocarcinogenesis were studied in female B6C3F1 mice previously treated with a single dose of N-nitroso-N-diethylamine (NDEA) at 7 days of age. The CCl4 treatments were given every other week and started either after weaning or at 18-26 weeks of age, and the animals were observed until 36 weeks of age. The number and size of hepatocellular nodules were then scored and analysed. Hepatocellular nodules were not observed in mice receiving either 4 or 8 CCl4 treatments without NDEA initiation. Both 4 and 8 CCl4 treatments promoted NDEA-initiated hepatocarcinogenesis, increasing nodule size but not nodule frequency. Eight CCl4 doses were more effective than 4 and gave the greatest increase in nodule size when administered from 18 to 32 weeks of age. In an additional experiment, 3 mice bearing hepatocellular nodules induced by a single infantile NDEA treatment were administered CCl4 once and killed 52 h later. Nine neoplastic nodules were examined and found to be resistant to the CCl4 induced necrosis, although cells in the nodules were actively dividing at the same rate as in the regenerating surrounding normal liver parenchyma. The ability of a few necrogenic CCl4 doses to enhance hepatocarcinogenesis may be related to a differential cytotoxicity on neoplastic and normal hepatocytes and to a similar mitogenic stimulus on both cell types.

Chromosome abnormalities and fragile sites in human melanoma

Chromosome analysis in short-term lines of three primary and seven metastatic malignant melanomas showed aneuploid karyotypes with recurrent abnormalities of chromosomes 1 (five cell lines), 6 (nine cell lines), and 7 (six cell lines). The breakpoints observed on the rearranged chromosomes frequently coincided with loci of known oncogenes and fragile sites. Two of the cell lines were analyzed after xenograft into nude mice and showed the presence of the same chromosomal changes observed in the parental cell lines, indicating the stability of the karyotype. A tendency toward an increased chromosomal fragility in peripheral blood lymphocytes was observed in five melanoma patients compared to ten normal individuals. However, there was no increased level of expression of specific fragile sites corresponding to the breakpoints observed in melanoma cells.

Comparison of urethane-induced sister-chromatid exchanges in various murine strains, and the effect of enzyme inducers.

The induction of sister-chromatid exchanges (SCEs) by urethane, 150 and 300 mg/kg administered i.p., was examined in bone-marrow cells of AKR, BALB/c, C3Hf, C57BL/6J and DBA/2 male mice. In all strains, the base-line level of SCE/cell was similar, ranging from 4.3 to 8.7, and the response increased with the dose of urethane. DBA/2 mice were the most susceptible to urethane at both dose levels, with 30.6 SCE/cell after treatment with 300 mg/kg, whereas the response of the other strains was from 17.4 to 21.5 SCE/cell at the same urethane dose. Pretreatment of C57BL/6J and DBA/2 mice with phenobarbital decreased the SCE frequencies induced by urethane, 300 mg/kg, to 70%, whereas a prior administration of beta-naphthoflavone reduced SCE levels in C57BL/6J but not in DBA/2 mice.

Two-Stage Liver Carcinogenesis in the Mouse:

A two-stage protocol for studying liver carcinogenesis was applied to the mouse. The protocol includes the treatment of 7-day-old mice with a single low dose of an initiating agent (diethylnitrosamine, NDEA), promotion starting after weaning and lasting about 20 weeks, and histologic analysis, at 30 weeks of age, of hepatocellular nodules onH&Estained sections. A stereologic analysis of results allows the evaluation of nodule frequency and size. Using this protocol in B6C3 and B6C mice, we have identified the promoting activity of the phenobarbital-like enzyme inducer, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), which was found to be a strong hyperplaseogenic agent for mouse liver. These studies also indicated that the different susceptibility to hepatocarcinogenesis in B6C3 and B6C mice may be related to a higher susceptibility of B6C3 than B6C initiated liver cells to growth stimulation. A long-term study showed that B6C mice have a low incidence of spontaneous liver tumors but are susceptible to chemical hepatocarcinogenesis and, therefore, they may be an alternative model to B6C3 mice in carcinogenesis bioassays.

Effects of phenobarbital and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene on differentiated functions in mouse liver

The promoters of murine hepatocarcinogenesis phenobarbital (PB) and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) given to adult C3Hf female mice increased the content of total liver DNA by 1.6-1.8-fold each week after the beginning of treatment. Both compounds increased the aminopyrine-N-demethylase activity, decreased the glucose 6-phosphatase (G6Pase), alkaline phosphodiesterase I and alkaline phosphatase specific activities, but did not modify the gamma-glutamyltransferase levels. Both compounds decreased the abundance of tyrosine aminotransferase- and metallothionein I-related RNA transcripts. These findings confirmed the PB-like activity of TCPOBOP and showed that both chemicals had a pleiotropic effect on mouse liver, that was not limited to stimulation of drug metabolism, but also affected other hepatocyte functions.

Kinetics of sister-chromatid exchange induction by different carcinogens in C57BL/6J and DBA/2 mice

Sister-chromatid exchange (SCE) levels were determined in bone marrow cells of DBA/2 and C57BL/6J mice at 18, 24, 30 and 48 h after treatment with 10 mg/kg cyclophosphamide (CP), 300 mg/kg urethane or 25 mg/kg N-nitroso-N-methylurea (NMU). DBA/2 mice showed higher SCE frequencies than C57BL/6J mice at all time points examined after CP and urethane treatment, whereas NMU administration resulted in similar SCE levels in both strains. After CP and urethane treatments, SCE values reached the highest level at 18 h, were similarly high at 24 and 30 h, and returned to base-line level at 48 h. In NMU-treated mice, the SCE values had the same level at 18, 24 and 30 h and returned to normal at 48 h. The results are interpreted as indicating that different metabolic capabilities are responsible for the difference in SCE response in the two strains.

Carcinogenicity study in mice on pildralazine, a hydralazinelike antihypertensive compound.

SummaryPildralazine, a hydralazinelike antihypertensive vasodilator containing a free hydrazine group, was administered in the drinking water to male and female B6C3F1 and to female BALB/c mice at 100, 200, and 400 ppm dose levels for 80 weeks. The animals were kept under observation until 130–133 weeks of age, when the experiment was terminated. A transplacentalinfantile bioassay was also carried out with pildralazine administered in the drinking water at 200 and 400 ppm dose levels to female C57BL/6J mice for 1 week before mating with C3Hf males and during mating and pregnancy. The progeny received the same doses for 10 weeks after birth and were kept under observation until 80–85 weeks of age, when the experiment was terminated. In both long-term and transplacental-infantile assays, control and treated groups developed the pattern of tumors usually observed in the strains used and no tumor type at any site appeared to be related to treatment.

Lack of carcinogenicity in mice of 4,4′-diaminobenzanilide and 4,4′-diaminoazobenzene, two intermediates used in the manufacture of azo dyes

4,4-Diaminobenzanilide and 4,4-diaminoazobenzene were administered in the diet to BALB/c mice at 100, 300 and 600 ppm dose levels for 60 weeks. The animals were kept under observation until 140 weeks of age when the experiment was terminated. Control and treated groups developed the pattern of tumours usually observed in BALB/c mice. No tumour type at any site was related to treatment.