S. D. Vesselinovitch

Carcinogenicity of diethylnitrosamine in newborn, infant, and adult mice

SummaryModifying effects of age, sex, and mouse strain on diethylnitrosamine (DEN) carcinogenesis have been investigated in C57BL/6Jx C3HeB/FeJ F1 (B6C3F1) and C3HeB/FeJxA/J F1 (C3AF1) hybrid mice. Animals each received four IP injections of 1.5 or 3.0 μg DEN/g body weight. The first injections were administered on days 1, 15, or 42 of life. Subsequent treatments were delivered at 3-, 6-, and 6-day intervals, respectively. Mice were kept under observation for the remaining life-span. DEN treatment induced tumors in liver, lungs, and forestomach in descending order of frequency. The majority of the induced liver tumors were hepatocellular carcinomas. Animals treated as newborns and infants developed significantly more liver tumors than animals that were treated as young adults. Newborn and infant females developed liver tumors at a later age (B6C3F1) and with a lower incidence (C3AF1) than similarly treated males. The B6C3F1 mice developed more hepatocellular carcinomas and a higher rate of pulmonary metastases than the C3AF1 mice. In contrast, C3AF1 mice developed lung tumors with a higher incidence and multiplicity than B6C3F1 hybrids. Forestomach tumors were observed also with a slightly but significantly higher incidence in C3AF1 mice.

Certain Aspects of Hepatocarcinogenesis in the Infant Mouse Model

The studies related to the kinetics of hepatocarcinogenesis and the effect of sex hormones upon hepatocarcinogenesis have been presented. The objective was to clarify the carcinogen- and time-dependent events leading to the development of liver tumors and the role of sex hormones regarding the rate of development of focal and nodular liver lesions. Kinetics of hepatocarcinogenesis were evaluated in B6C3F1 mice using the mathematical-graphic approach. Low-dose levels (0.0, 0.3125, 0.625, 1.25, 2.5, and 5.0 μg/g body weight) of diethylnitrosamine (DEN) were injected once intraperitoneally into 15-day-old males. Subgroups of 8 to 20 animals were killed from each treatment level at 4 to 6 week intervals. In addition, a series of male and female mice was administered 2.5 μg DEN/kg body weight and a fraction of each sex was gonadectomized. The analysis of dose-response data showed first order kinetics (single event) regarding the induction of intermediate basophilic foci (IBF) and hepatocellular carcinomas (HCC). The difference in the transformation probabilities between these two lesions was of 3 orders of magnitude, indicating a qualitative difference between the original events. Time-dose kinetics showed that twice as many time-dependent events were required for the development of HCC than for the development of IBF. Therefore, the carcinogen influenced not only the nature and degree of initiation of hepatocytes but also programmed the rate of cascading events, and the time being the limiting factor of morphologic expression. In relation to the role of sex hormones, the study demonstrated that the male hormonal environment accelerated and the female hormonal environment delayed the rate of focal morphologic expression and neoplastic progression.

Liver Tumor Induction.

The significance of the development of nodular liver lesions in rodents following the administration of test agents raises several questions which could be placed in one of two general categories: diagnostic and interpretational. From a diagnostic point of view, the proper classification of liver tumors into a benign and malignant category has to be based on the direct correlation between the morphology and the biologic behavior of the lesions. Therefore, extreme care should be taken to separate the malignant tumors from the benign and the benign neoplasia from the hyperplasia. The substitution of the term “neoplastic nodule” for hyperplastic nodule in rats is misleading. Most of these nodules, when induced under special experimental conditions, may regress or remodel and thus they are not neoplastic in nature. Chronic carcinogenicity bioassays should include “stop” type of treatment leaving enough of the observational time to establish the fate of induced nodular lesions. The induction of histochemically changed foci can serve only as an indication of potential hepatocarcinogenicity and should not be equated with the induction of bona fide cancer. The biologic interpretation of nodular liver lesions, especially in mice, needs further scrutiny because these lesions have a tendency to develop spontaneously with high incidence in some strains. This characteristic then raises the question as to the mechanism by which various agents augment and/or accelerate the development of such tumors. Is this action primarily promoting or initiating in nature or does it represent the induction of tumors de novo? The answer to this dilemma may have a decisive bearing on carcinogenic risk assessment and the type of regulatory action, since the promoting agents possess a threshold effect and the promoted changes may regress following withdrawal of treatment. The interpretation of hepatocarcinogenesis is further complicated by the fact that several factors, such as sex hormonal environment, increased mitotic activity following an excessive loss of parenchymal cells, degree of caloric intake, enzymatic complement, and animals’ age at the time of the exposure to a test agent, may influence the outcome of liver tumor development by modulating “initiation” and/or “promotion” of carcinogenesis. Broad fluctuation in the historic incidence of liver tumors further compounds the complexity of the proper bioassay interpretation. The specifically designed experiments may have the objective to explore predominantly the initiating or promoting effects of the agent. Such protocols should be used whenever necessary to differentiate between these two mechanisms of action. In the Caucasians, the “spontaneous” development of the primary hepatocellular tumors is rare. The majority of these tumors are malignant and rapidly fatal. According to some human pathologists, the benign variety of liver tumors is rare and it does not represent necessarily a premalignant stage in tumor development. Carcinoma of the liver may occur in infancy, especially in males before the age of 2 years. This suggests a genetic causation or carcinogenic exposure in utero. One of the geographic factors which significantly enhances the incidence of hepatocellular carcinoma in humans is exposure to aflatoxin B1 which is apparently potentiated by concurrent liver cirrhosis. Because many more agents have been found to be hepatocarcinogenic in mice and rats than in men, a question arises as to the direct relevance of rodent studies to humans. A balanced assessment of the carcinogenicity of the agent could only be reached in considering both the pharmacokinetics and the development of malignant neoplasia in other organs. In the case of positive carcinogenicity assessment, the outcome of the mutagenicity bioassays can suggest genic (genotoxic) or paragenic (epigenetic) mode of action in mammalian systems.

Induction of Focal and Nodular Liver Lesions in Rodents as an Indication of Human Carcinogenic Riska

We are gathered here to probe the mysteries of cancer, interpret their signs and, for some of us, to follow in the footsteps of the Roman augurs and the Etruscan haruspices to foresee the future regarding the carcinogenic risks to humans from naturally occumng and man-made agents. Because we are experimental pathologists, our primary task as haruspices belongs to the so-called exta category, according to which the future could be predicted after the examination of the internal organs, primarily the liver, regarding their size, color, shape, and various markings. These omens were interpreted according to the well-established criteria developed in Piacenza, close to Bologna, which further justifies the discussion of the topic.

Sex Hormones as Modulators of Liver Tumor Development

The induction of hepatocellular tumors depends upon the nature of the chemical agent (1), its requirement for activation to ultimate carcinogenic moiety (2), enzymatic competence of tissue(s) to activate the procarcinogen (3–5), the interaction of ultimate carcinogenic moiety with specific macromolecular site(s) (6,7), the rate of removal of the formed adduct(s) (8), the degree of macromolecular repair (9,10), caloric intake (11–13), immune competence, and hormonal environment of the host (14–17), The interplay of all these factors contribute in varying degrees to the neoplastic expression in the liver. The species, strain (18), age at treatment (19), and sex (19) of the animals may influence the degree of the agent’s activation. In addition, the age at treatment may affect the macromolecular damage-repair ratio and consequently the degree of fixation of the residual macromolecular lesion(s) (20).

Histochemical Characteristics of Foci Induced by Diethylnitrosamine, Liver, Mouse

The process of hepatocarcinogenesis has been studied extensively in rats with the objective of identifying progenitors of the sequentially emerging cell populations (Bannasch et al. 1980; Emmelot and Scherer 1980; Farber 1980; Pitot and Sirica 1980; Williams 1980). Studies, much more limited in scope, have been carried out in mice (Becker and Sell 1979; Becker et al. 1977; Butler and Hempsall 1978; Jalanko and Ruoslahti 1979; Lipsky et al. 1981; Moore et al. 1981; Vesse-linovitch 1980; Vesselinovitch et al. 1979, 1982, 1984; Vesselinovitch and Mihailovich 1983 a, b). The objective of the present study was to investigate the enzyme histochemical pattern of the focal lesions identified as basophilic foci in hematoxylin and eosin stained sections within the first 24 weeks of treatment with diethylnitrosamine (DEN). The study focused on the enzymes and metabolic products related to carbohydrates.