Histochemical profile of mouse hepatocellular adenomas and carcinomas induced by a single dose of diethylnitrosamine.

In continuation of earlier studies on murine neoplastic liver lesions, we characterized by histochemical methods the phenotype of hepatocellular adenomas and carcinomas induced by single injections of diethylnitrosamine (1.25, 2.5, or 5.0 micrograms/g of body weight) in 15-day-old C57BL/6 x male C3H F1 mice. The hepatocellular adenomas were composed predominantly of basophilic cells but stored excessive amounts of fat and glycogen in large portions of the tumors. Irrespective of the carcinogenic dose, the adenomas showed a consistent histochemical pattern. Glycogen synthase and phosphorylase were highly active in the hepatocytes that stored glycogen. In cells poor in, or free of, this polysaccharide, these enzymes were only moderately active or even inactive. In glycogen-storing parts of the adenomas, the activity of adenylate cyclase was reduced compared with normal liver parenchyma, but in fat-storing portions it was elevated. In a few adenomas, uniform increase in adenylate cyclase activity could be encountered. The levels of ATPase, acid phosphatase, and glucose-6-phosphatase were either increased or decreased. Glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase showed an increased activity in all adenomas compared with preneoplastic foci, which in turn exhibited a higher glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase activity than the surrounding parenchyma or the liver of untreated controls. The hepatocellular carcinomas showed remarkable histochemical changes compared with adenomas. The levels of fat and glycogen and the activities of glycogen synthase, phosphorylase, and in most cases also that of glucose-6-phosphate dehydrogenase, were reduced significantly. In contrast, adenylate cyclase, glucose-6-phosphatase, glyceraldehyde-3-phosphate dehydrogenase, and also alkaline phosphatase showed a striking elevation in developing carcinomas. Similar, although more pronounced, histochemical changes were seen in the advanced hepatocellular carcinomas. These observations indicated that progression from adenomas to hepatocellular carcinomas was associated with a change in the activity of several enzymes involved in cell membrane function, glycogen metabolism, the oxidative pentose phosphate pathway, and glycolysis.

Comparative developmental and phenotypic properties of altered hepatocyte foci and hepatic tumors in rats.

Previous investigations in this laboratory have provided evidence that histochemically detectable altered hepatocyte foci and hepatic tumors appearing in rats given a single neonatal treatment with a low dose of carcinogen followed by chronic dietary phenobarbital administration are developmentally independent. The present investigation further evaluates developmental relationships among these lesions. Altered hepatocyte foci were divided into two subclasses consisting of foci that were detectable by histochemical as well as by hematoxylin-eosin staining [designated hist(+)/morph(+) foci] and those foci that were detectable solely by histochemical staining [designated hist(+)/morph(-) foci]. The developmental and phenotypic properties of the hist(+)/morph(-) foci, hist(+)/morph(+) foci, and hepatic tumors were compared in rats initiated once neonatally with different doses of diethylnitrosamine and promoted with dietary phenobarbital from weaning. The morph(+) and morph(-) lesion subclasses were distinguishable on the basis of several developmental characteristics. Hist(+)/morph(+) foci were present at low frequency until at least 150 days after initiation. Although the development of hist(+)/morph(-) foci was essentially complete at that point, the rate of appearance of hist(+)/morph(+) increased significantly. The diethylnitrosamine dose response of the hist(+)/morph(+) foci followed the histochemical marker patterns of the tumor lesion class more closely than that of the hist(+)/morph(-) group. The rates of expression of the hist(+)/morph(+) foci increased with the increasing level of histochemical complexity, whereas the rates of expression of the hist(+)/morph(-) foci groups were inversely correlated to their complexity level. Although the average focus size or diameter in the hist(+)/morph(+) groups was greater than that of the hist(+)/morph(-) foci, the focus growth rates of morph(+) and morph(-) subsets matched for histochemical phenotype were comparable. The complexity level and individual marker distribution patterns of the hist(+)/morph(+) focus class were more similar to tumor patterns than to the distribution patterns of the hist(+)/morph(-) lesion class. The results suggest the following. (a) The development of lesion classes with successively greater deviation from normalcy does not occur via lineal progression from less to more deviated forms within a given lesion class. The three lesion classes appear to develop independently, with the developmental characteristics of each lesion class determined at the time of initiation.(ABSTRACT TRUNCATED AT 400 WORDS)

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 micrograms/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 micrograms 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.

Invasiveness, metastasis, and transplantability of mouse liver nodules.

Data are presented which show that various mouse liver nodular lesions, such as hyperplastic nodules, hepatocellular adenomas, and hepatocellular carcinomas, differ not only morphologically but also biologically regarding their behavior in the primary and secondary hosts. The hyperplastic nodules grew slowly by expansion without affecting the integrity of the lobular structure. The primary hepatocellular adenomas also grew slowly by expansion but eliminated the histoid liver structure. Neither lesions metastasized to the regional lymph nodes and distant organs. Upon isogeneic transplantation, the adenomas required a long latent period before manifesting an expansive type of growth without invasion and metastasis. The primary hepatocellular carcinomas grew fast, destroyed surrounding parenchyma, and those showing moderately to poorly differentiated cellular morphology metastasized to regional lymph nodes and distant organs. Upon transplantation hepatocellular carcinomas grew fast, invaded locally, metastasized distantly, and killed the recipients within a few weeks. Thus hepatocellular adenomas and carcinomas represent two distinct entities both morphologically and biologically. Thus the agents should be classified according to the type of nodular liver lesions they "induce" in the mouse liver. The least innocuous agents would be those resulting in the development of hyperplastic nodules. They should not be labelled as hepatocarcinogens. On the other side of the spectrum should be the agents "inducing" the hepatocellular carcinomas and they should be classified as mouse liver carcinogens. The agents capable of affecting significantly the hepatocellular adenomas but not the hepatocellular carcinomas should be considered as tumorigens, since they are causally related only with the benign liver lesions. Combining both benign and malignant liver tumors for statistical purposes is not justified unless each type of tumor has been affected in a statistically significant incidence. If this were not the case, classification of the agent should be in accordance with the type of lesions induced significantly. In order to use the induction of hepatocellular carcinomas in rodents as an indication of carcinogenic risk in humans appropriately, one has to verify within the limits of feasibility that the identical carcinogenic effects and the subsequent events occur both in rodents and humans.(ABSTRACT TRUNCATED AT 400 WORDS)

Oncogenic evaluation of tetrachlorvinphos in the B6C3F1 mouse.

Groups of 80 male and 80 female B6C3F1 mice were fed diets containing 17.5, 64, 320, 1600, 8000, and 16000 ppm tetrachlorvinphos (TCVP) for up to 103 weeks. Another group of 80 male and 80 female mice were fed TCVP (16000 ppm) that was used in a previous bioassay. One hundred-sixty male and 160 female mice served as the control group. Ten treated and 20 control mice/sex/group were killed at 6, 12, and 18 months. It was estimated that the study maximum-tolerated dose was exceeded by three- and sixfold in the 8000- and 16000-ppm dose groups, respectively. Consequently, these exposures produced excessive cytotoxicity and regenerative changes in the liver and kidneys which were associated with sex-hormonal imbalance and metabolic overload in liver. A significant decrease (15-40%) in body weight was observed in mice fed 8000 and 16000 ppm TCVP. These treated mice did not gain weight during the study. Reduced food consumption and caloric intake throughout the study were probably responsible for the increased survival and the decreased incidence of spontaneous neoplasia in mice fed 8000 and 16000 ppm TCVP. Classification of pathologic lesions observed in these high-dose groups differed among study and consulting pathologists. The consultant and Shell pathologists concluded that the liver and kidney changes were causally related to excessive toxicity which was manifest primarily by hepatocellular hyperplasia and renal tubular adenoma. Study pathologist in accordance with his classification found statistically significant increases in hepatocellular carcinoma, hepatocellular adenoma or carcinoma, and renal tubular carcinoma in male mice fed 16000 ppm TCVP. The incidence of hepatic neoplasms as evaluated by the study pathologist in female mice fed 8000 and 16000 ppm TCVP although statistically significant was of questionable biologic significance when compared with historical female controls. The only statistically significant finding observed by the consulting pathologist was an increased incidence of renal tubular adenoma and renal tubular adenoma or carcinoma in male mice fed 16000 ppm TCVP. Use of results from these high-dose groups is contraindicated due to the many compromising factors affecting mice fed 8000 and 16000 ppm TCVP. TCVP was found not to be oncogenic in B6C3F1 mice at dose levels not exceeding the maximum tolerated dose.

Histochemical characterization of focal hepatic lesions induced by single diethylnitrosamine treatment in infant mice.

Focal hepatocellular lesions, induced in our infant mouse system (15-day-old B6C3F1 mice) by a single carcinogenic dose of diethylnitrosamine (2.5 or 5.0 micrograms/g body weight), were characterized histochemically using toluidine blue, periodic acid-Schiff, glycogen phosphorylase, glycogen synthetase, glucose-6-phosphatase, glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, ATPase, gamma-glutamyl transpeptidase, and acid phosphatase. Animals were killed 5, 12, 18, and 24 weeks following diethylnitrosamine treatment. The first focal lesions were observed in mice killed at 12 weeks. All foci showed patchy cytoplasmic basophilia and a slight decrease in the glycogen content. The early foci (12 weeks) showed no change in the levels of glycogen phosphorylase and glycogen synthetase, a strong reduction of glucose-6-phosphatase, and a high increase in glucose-6-phosphate dehydrogenase. In addition, 56% of foci in males and 86% of foci in females showed a slight rise in glyceraldehyde-3-phosphate dehydrogenase, and 12% of foci in males and 17% of foci in females had a lower acid phosphatase. The level of cytoplasmic ATPase was slightly decreased in 22% of foci. By 24 weeks, a decrease in the activity of cytoplasmic ATPase was observed in 84 and 100% of foci in males and females, respectively. The increase in the membrane ATPase was observed in 65% of foci in males and 7% of foci in females. By that time, the decrease in acid phosphatase was observed in 78% of foci in males and 37% of foci in females. The gamma-glutamyl transpeptidase failed to show any increase in its activity, indicating that this enzyme was not a "marker" of the hepatocellular lesions developing under the experimental conditions. Strong decrease in glucose-6-phosphatase in association with a manifest increase in glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase activities indicated a shift from gluconeogenesis to glycolysis. Since this metabolic shift occurred concurrently with an increase in the labeling indices and focal size, it appears that these changes act in concert, representing expression of the acquired functional and replicating potential of the focal cell population.

Characterization of histochemically detectable altered hepatocyte foci and their relationship to hepatic tumorigenesis in rats treated once with diethylnitrosamine or benzo(a)pyrene within one day after birth.

A new experimental system was used to examine the stages of chemically induced hepatic neoplasia in the rat. The treatment protocol involved the i.p. injection of a single dose of carcinogen [diethylnitrosamine or benzo(a)pyrene] into male and female rats within 1 day after birth, followed by dietary exposure to promoter (0.05% dietary phenobarbital) beginning at weaning. Rats were killed at intervals, and their livers were examined for tumors and for histochemically detectable foci of altered hepatocytes using six histochemical markers. Through serial frozen-sectioning techniques and computer-assisted image analysis, foci containing between one and six markers were identified, and their average diameters were calculated. The same complement of histochemical tests was applied to the primary hepatic tumors observed in this study. The principal findings were the following. (a) Both the diethylnitrosamine and benzo(a)pyrene treatments were tumorigenic and produced foci with similar phenotypic properties (numbers and identities of histochemical markers). (b) Foci relative growth rates and growth capacities (ranges of possible growth rates) were directly related to foci phenotypic complexity levels (numbers of markers per focus). (c) Individual foci were phenotypically stable; i.e., they neither gained nor lost markers. (d) A substantial fraction of the tumors observed in this study had fewer markers than the most complex foci. On the basis of these observations, we suggest that foci emerge as the result of a specific set of cellular changes solely inducible by carcinogenic stimuli, but the foci do not evolve through progressively more deviated forms into tumors. Instead, we postulate that tumorigenesis involves a separate transformation event that may occur in a susceptible fraction of foci subsequent to their induction or, alternatively, may occur at the time of exposure to carcinogen, in parallel with the carcinogen-mediated events leading to focus formation.

Relationship of histochemically detectable altered hepatocyte foci to hepatic tumorigenesis.

A new experimental system was used to examine the stages of chemically induced hepatic neoplasia in the rat. The treatment protocol involved the intraperitoneal injection of a single non-necrogenic dose of carcinogen [N-nitrosodiethylamine (NDEA) or benzo[a]pyrene (BP)] into male and female rats within one day after birth, followed by dietary exposure to promoter (0.05% phenobarbital) from weaning. Rats were killed at intervals, and their livers were examined for tumours and for histochemically detectable foci of altered hepatocytes. Six histochemical markers were used. Through serial frozen-sectioning techniques and computer-assisted image analysis, foci containing between one and six markers were identified, and their average sizes were calculated. The same complement of histochemical tests was applied to the primary hepatic tumours observed in this study. The data showed that (1) the new treatment protocol was highly efficient in foci and tumour production; (2) growth rates and incidence levels of foci were directly related to hepatocarcinogenic effectiveness (NDEA greater than BP), whereas both carcinogens had similar effects on foci phenotypic properties; (3) after their formation, foci at a given level of phenotypic complexity did not become progressively more complex; (4) incidence levels of foci were sex-dependent (females greater than males), but growth rates of foci were the same for both sexes; (5) growth rates and growth capacities (ranges of possible growth rates) of foci were directly related to phenotypic complexity levels of foci; (6) frequencies and phenotypic complexities of foci were inversely related; the reverse was true for tumours, although 10% of the tumours were relatively simple (three markers or fewer); (7) marker frequency distribution patterns were completely different in foci and in tumours. From these observations, we suggest that the foci are not direct tumour progenitors but, instead, are manifestations of a mosaic of subtumorigenic effects of the carcinogenic stimulus on cellular functions associated with the control of cell division and phenotypic character. The observed foci and tumour characteristics suggest that these carcinogen-induced cellular changes define elements of the mechanism whereby a specific neoplastic transformation site is rendered more accessible to carcinogenic attack.

Carcinogenicity of diethylnitrosamine in newborn, infant, and adult mice.

Modifying 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 micrograms 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.

Kinetics of diethylnitrosamine hepatocarcinogenesis in the infant mouse.

Kinetics of hepatocarcinogenesis was evaluated in 15-day-old male C57BL/6J X C3HeB/FeJ F1 mice using a nontoxic carcinogenic dose range of diethylnitrosamine (DEN). The carcinogen was injected i.p. once, and the animals were killed according to the protocol. Two studies were carried out sequentially over a period of 4 years. In the first study, groups of mice were treated with 0.625, 1.25, 2.5, and 5.0 micrograms of DEN per g of body weight, and subgroups of eight mice were killed at 10-week intervals, the first at 10 weeks following carcinogenic treatment. The dose-response relationship, transformation probabilities, and the dose versus time to 50% incidence of the early (basophilic foci) and later appearing focal and nodular hepatocellular lesions were evaluated. In the second study, groups of mice were treated with 0.312, 0.625, 1.25, 2.5, and 5.0 micrograms of DEN per g of body weight, and subgroups of 8 to 20 animals were killed at 10, 16, 20, 24, 30, 34, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, and 110 weeks after carcinogenic treatment. The numbers of induced basophilic foci and hepatocellular carcinomas per number of liver cells at risk (transformation probabilities) were used to evaluate dose-response, time-response, and time-dose kinetics. In both studies, the kinetics of hepatocarcinogenesis was evaluated from data plotted on the double logarithmic scale. Regardless of the dose used, DEN induced four distinct morphological entities: basophilic (glucose-6-phosphatase deficient) foci; hyperplastic nodules; hepatocellular adenomas; and hepatocellular carcinomas in all animals. The first study demonstrated a positive dose-response relationship and constancy (k) of the product of single dose (d) and the time to 50% (t50%) incidence (d . tn50% = k) for each of the four morphological entities. The numerical value of the power of time (n) increased from 2.6 to 2.7, 3.4, and 5.7 for the above four lesions, respectively. The second study showed first-order kinetics regarding the induction of basophilic foci and hepatocellular carcinomas. The transformation probability of development of basophilic foci was up to three orders of magnitude greater than that observed for development of hepatocellular carcinomas, suggesting a qualitative difference between these two types of hits. The time-response kinetics showed that the development of basophilic foci and carcinomas was related to the time factor by powers of 2 and 4 for these two lesions, respectively. The time-dose relationship to a fixed number of lesions per number of liver cells at carcinogenic risk showed a negative slope with an n value of 2 for the induction of basophilic foci (d . t28/10(7) = k) and an n value of 4 for the induction of hepatocellular carcinomas (d . t40.08/10(7) = k). The data indicated that at least two critical events are needed for the induction of basophilic foci and at least four events are required for the induction of hepatocellular carcinomas...

Lindane bioassay studies and human cancer risk assessment.

Benzene hexachloride or hexachlorocyclohexane comes in eight isomeric forms which differ in the spatial positions of the chlorine atoms on the boat and chair forms. Only one, however, gamma-isomer (lindane) possesses relevant insecticidal activity. The majority of studies on the potential carcinogenicity of benzene hexachloride, however, did not discriminate between the various isomers because they assumed that carcinogenicity is independent of steric structure. Also, proper differentiation between morphologic endpoints was not duly carried out and considered. The present evaluation of lindane's biologic activity was restricted to the lifelong studies in which pure lindane (greater than 99.5%) was utilized and the morphology of the observed lesions, mainly in the liver, were classified according to their biologic behavior into hyperplastic, benign neoplastic, and malignant entities. None of the studies gave evidence that lindane was carcinogenic in mice and rats. An enhancement of benign liver tumors (tumorigenic effect) was observed only in CF1 male mice (17/56 at the only tested dose: 400 ppm vs. 3/89 observed in the nontreated controls). Dose-response function of this tumor response in conjunction with two other lifetime studies (80 and 160 ppm in B6C3F1 and 50, 25, and 12.5 ppm in NMRI mice) were best described by the Weibull model, giving a relatively high estimate of its shape parameter (m = 2.5). Assuming similar susceptibility of humans to lindane and considering maximal occupational exposure to this agent (professional applicators), the probability of lindane associated tumor development was estimated as 2.2 X 10(-10). Because this cancer risk is five orders of magnitude below the acceptable health risk, this evaluation concludes that environmental exposure to lindane does not pose a threat to human health.

Perinatal hepatocarcinogenesis.

The mouse species is biologically characterized by a short gestation period that precludes completion of the intrauterine development before birth. Thus, in the mouse the late phase of intrauterine development is carried over into the postnatal age period. For this reason, we studied chemically induced hepatocarcinogenesis in B6C3F1 mice that were exposed transplacentally, postnatally, or during adulthood to benzidine x 2HCl, safrole, amitrol, ethylnitrosourea (ENU), and diethylnitrosamine (DEN). Data showed that perinatal exposure to those agents were significantly more hepatocarcinogenic than similar adult exposure. As regards the perinatally treated animals, the infants were shown to be more responsive than fetuses. The discussion shows that the higher "susceptibility" of infants was causally related to the presence of enzymatic complement required for the activation of procarcinogens and the concurrent high rate of macromolecular replication. It has been concluded that carcinogenesis in infants may be viewed as a part of the late "intrauterine" development. Thus, observed carcinogenicity has been construed as being relevant to the potential risk that might occur in other species exposed to these carcinogens during the late phase of intrauterine life.

Infant mouse as a sensitive bioassay system for carcinogenicity of N-nitroso compounds.

Five groups of 48, C57BLxC3H F1, male mice, 15 days old, were administered NDEA intraperitoneally at the following levels: 0.0 (saline only), 0.625, 1.25, 2.50 and 5.00 microgram per g body weight, in a single dose. Groups of eight mice from each of the dose levels were killed at 40, 50, 60, 70, 80 and 90 weeks of age. The nodular liver lesions were classified s (a) focal areas of non-specific cellular changes, (b) hyperplastic nodules, (c) hepatocellular adenomas and (d) hepatocellular carcinomas. Regardless of the dose, all the animals developed hepatocellular carcinomas. The average latent periods, however, were inversely proportional to dose, being 66 weeks with the lowest dose and 44 weeks with the highest dose. The multiplicity and the average weight of the early nodular lesions (50 weeks) was directly related to th NDEA dose. Thus the higher multiplicity was associated with faster emergence of hepatocellular carcinomas. The high susceptibility to hepatocellular carcinogenesis, in association with the short latency, makes the infant mouse a sensitive bioassay system for assessing the carcinogenic potential of N-nitroso compounds.

Opium: a potential urinary bladder carcinogen in man.

Evidence is presented that opium addition is a risk factor for cancer of the bladder. A case-control study of 99 bladder cancer patients admitted to Nemazee Hospital in Shiraz, Iran was evaluated. Cancer patients and controls, matched by age and sex, were analyzed as to their opium and/or cigarette smoking habits. A high correlation between opium addiction and bladder cancer has been observed. This evidence significantly exceeded the one observed in relation to cigarette smoking only. The sex ratio for this cancer site, from hospitalized cases in Southern Iran, is estimated to be about nine male cases per one female case. This high male to female ratio was attributed to the greater addiction of males to opium. It was concluded that opium and, more likely, its pyrolysis derived fractions may represent potential bladder carcinogens in man.