Mouse Strain Susceptibility to Diethylnitrosamine Induced Hepatocarcinogenesis Is Cell Autonomous Whereas Sex‐susceptibility Is Due to the Micro‐environment: Analysis with C3H ↔ BALB/c Sexually Chimeric Mice

Abstract

In man, liver cancer is on the increase, especially in males. Sex differences also exist in rodent models. To elucidate the mechanisms, chimeric mice were produced by amalgamation of early embryos from high and low hepatocarcinogen-susceptible strains, C3H and BALB/c. Tumor formation was initiated with 10 mg/kg of diethylnitrosamine at the ages of 7 and 14 days and mice were sacrificed at 30 and 45 weeks. The chimeras were classified into XY ↔ ↔ ↔ ↔XY, XY ↔ ↔ ↔ ↔XX, XX ↔ ↔ ↔ ↔XY, and XX ↔ ↔ ↔ ↔XX in terms of sex chromosomes by means of polymerase chain reaction-simple sequence length polymorphism analysis (SSLP) using Y chromosome-specific Sry primers in combination with the D3Mit21 marker. Liver lesions were analyzed histopathologically, by immunostaining using a C3H strain-specific antibody and by DNA in situ hybridization with the Y chromosomespecific digoxigenin-labeled Y353/B probe. Sex and strain genotyping by SSLP analysis matched histological observations, confirming the reliability of our system. The strain differences in liver tumor numbers of each strain type in XY↔ ↔ ↔ ↔XY and XX ↔ ↔ ↔ ↔XX subtypes of C3H↔ ↔ ↔ ↔BALB/c chimeras were retained well (P < < < <0.0001 and P < < < <0.001, respectively), indicating a minimum influence of the C3H or BALB/c surrounding milieu on development of individual lesions. On the other hand, significant promotion of XX cell tumors was evident in phenotypically male sexually chimeric XY ↔ ↔ ↔ ↔XX and XX ↔ ↔ ↔ ↔XY chimeras for both C3H (P < < < <0.02) and BALB/c (P < < < <0.01) lesions compared to the XX ↔ ↔ ↔ ↔XX case. The results suggest the presence of hormonal or micro-environmental factors specific for males, which are not caused cell-autonomously. Basic strain differences, however, are determined by intrinsic genetic factors rather than the strain-dependent micro-environment.Key words: Chimera -Hepatocarcinogenesis -Strain susceptibilityIn man, the occurrence of primary liver cancer has been on the increase for several years, changes in rates being more marked in males than in females. 1) In Japan, 1996 death rates for intrahepatic neoplasia were 37.5 and 14.6/ 100 000 for males and females, respectively, according to the National Health and Welfare Statistics. 2)In rodent models, there also are sex differences in spontaneous and carcinogen-induced hepatocarcinogenesis, males being more susceptible than females.3, 4) One fundamental question is whether the sex differences are cellautonomous or caused by hormonal or micro-environmental factors. To address this issue, Kemp et al. 5) utilized Tfm (testicular feminization) mutant mice and showed that liver tumor promotion by testosterone required a functional androgen receptor in the intact animal. Other researchers reported that castration of males reduced the incidence of carcinomas and adenomatous nodules in 3′ ′ ′ ′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB)-treated 6) and in N,N-diethylnitrosamine (DEN)-treated mice.7) The results with DEN-initiated and 2-acetylaminofluorenetreated rats appear similar. 8) Furth...

The beneficial effect of natural antioxidants from olive oil with fig and date palm fruit extracts on biochemical and hematological parameters in rats during diethylnitrosamine-induced carcinogenesis

“…This indicates that DEN-induced oxidative stress might alter the hepatic lipid metabolism (Kim et al 2006;Fathy et al 2017); therefore, it is suggested that oxidative stress might be an important determinant of altered lipid metabolism from DEN treatment. Our results are also in accordance with those of Verna et al (1996) and Tsukamoto et al (2000) who reported an increase in lipids in the plasma of rats after DEN treatment, and attributed the hypercholesterolemia conditions to the stimulation of cholesterol synthesis in the liver after DEN treatment and its release from tissues due to destruction of cell membranes, and the mobilization of fats from the adipose tissues into the bloodstream in addition to mitochondrial dysfunction. Moreover, Bok et al (1999) attributed hypercholesterolemia to increased activation of 3-Hydroxyl-3-methyl glutaryl coenzyme A (HMG-CoA) reductase enzyme, the key regulatory enzyme in the reduction of the overall process of cholesterol synthesis.…”

Biological Basis of Differential Susceptibility to Hepatocarcinogenesis among Mouse Strains

“…While basic strain differences in hepatocarcinogen sensitivity are determined by intrinsic genetic factors, studies of C3H–BALB/c sexually chimeric mice treated neonatally with diethylnitrosamine show that male specific hormonal or micro-environmental factors are responsible for promotion of liver cancer in both XX and XY hepatocytes 135 .…”

“…As an indication of the magnitude of the hormonal effect, neonatal urethane exposure followed by castration or ovariectomy at 6 weeks of age resulted in a 96% and 20% incidence of heptomas in sham-operated male and female B6C3F1 hybrids, respectively, while orchidectomy and ovariectomy hybrids had hepatoma incidences of 62% and 67%, respectively 187 . Using C3H–BALB/c sexually chimeric mice, Tsukamoto and co-workers show that while basic strain differences are genetically determined, male hormonal or micro-environmental factors lead to promotion of liver cancer in both XX and XY hepatocytes 135 .…”

Diethylnitrosamine causes pituitary damage, disturbs hormone levels, and reduces sexual dimorphism of certain liver functions in the rat.

“…Spontaneous liver cancer in both humans and animals occurs predominantly in males (14)(15)(16)(17)(18)(19). Also, liver cancer induced in various experimental animal models usually shows a male predominance (14,17), as exemplified by the resistant hepatocyte model (20,21).…”

“…Also, liver cancer induced in various experimental animal models usually shows a male predominance (14,17), as exemplified by the resistant hepatocyte model (20,21). Several studies have shown that liver cancer formation induced by chemicals in animals can be affected greatly by various hormonal manipulations, such as hypophysectomy and castration, demonstrating the importance of hormones in the carcinogenic process (14,17,(19)(20)(21). In addition, administration of some carcinogens has been shown to influence the hormonal environment and the sensitivity of tissues to hormones.…”