Delayed effects of tamoxifen in hepatocarcinogenesis-resistant Fischer 344 rats as compared with susceptible strains.

The anti-oestrogenic drug tamoxifen has been under investigation as a breast cancer chemopreventive agent for at least a decade. However, its use for this purpose is still debatable since it is able to induce liver tumours in rats via a mechanism involving metabolic activation to a DNA adduct-forming electrophilic intermediate. The metabolic activation and adduct-forming properties of tamoxifen are now well characterized but less is known about its ability to induce hepatic cell proliferation, which is also essential for the carcinogenic process. The effects of tamoxifen on liver weight and cell proliferation were compared in female Fischer 344 (F344), Wistar and Lewis rats given the drug in the diet for up to 26 weeks. The onset and duration of hepatic cell proliferation varied between the strains of rat. In Wistar and Lewis but not F344 rats there was a marked increase in hepatocellular proliferation during the first 4 weeks of tamoxifen administration. In the Wistar strain this was associated with an increase in DNA adduct levels; no such increase was observed in the F344 strain. The onset of the proliferative response was delayed until the 13 week time point in the F344 strain. By the 13 and 26 week time points, cell proliferation in tamoxifen-treated Wistar and Lewis rat liver had returned to normal, but the amount of apoptotic activity in these livers was elevated. This suggests that excess cells generated during the proliferative phase of tamoxifen treatment were being eliminated by apoptosis. In the F344 strain, however, increased proliferative activity was associated with relatively low apoptotic activity at the 26 week time point, suggesting that the delayed proliferative response had yet to be balanced by apoptotic deletion. This is consistent with the fact that tamoxifen-induced hepatocellular tumours develop very late, towards the end of the lifespan, in this strain. The cell proliferative activity of tamoxifen in the Wistar rat liver was compared with that of a non-mutagenic analogue, toremifene. Tamoxifen induced increased cell cycle activity in the livers of rats following gavage dosing at all sampling times (1-12 weeks), whereas toremifene had no effect on the incidence of cycling in hepatic cells, demonstrating that the hepatic cell proliferation is not a general response to anti-oestrogen treatment. These observations suggest that the rate of promotion of liver tumours by tamoxifen is a function of the rate, time of onset and duration of increased cell replication. The susceptibility of rat strains to the hepatocarcinogenic effects of tamoxifen appears to depend upon the balance between initiation via DNA adduct formation, promotion via increased cell proliferation and cell deletion via apoptosis. Our findings suggest that an early proliferative response to tamoxifen is important in this process.

Mutations associated with in vivo aflatoxin B1-induced carcinogenesis need not be present in the in vitro transformations by this toxin.

Ingestion of aflatoxin B1 is implicated in the high incidence of human liver cancers in several developing countries. An association has been detected between human exposure to aflatoxins, and mutations in the third base of codon 249 of the p53 gene in hepatomas. In vitro experiments using human cell line cells and aflatoxin B1 have demonstrated the induction of p53 mutations in codon 249 and adjacent codons. It was therefore of interest to see if this correlation between the in vivo and in vitro situations held for other species. The present study examined a rat liver-derived cell line, transformed in vitro with aflatoxin B1, for the presence of mutations associated with in vivo aflatoxin-induced hepatocarcinogenesis. In an in vivo rodent model systems using the aflatoxin B1-sensitive male F344 rat, previous studies have shown that hepatocarcinogenesis is accompanied by significant incidences of codon 12 mutations in K-ras and codon 13 mutations in N-ras genes, but in contrast to the human, apparently not by mutations in codon 243 of the p53 gene (which corresponds to codon 249 in the human gene). In contrast to the situation in humans, mutation in the third base of codon 243 in the rat would not result in any changes in amino acid sequence, but mutations in codon 250, as seen in in vitro human systems, would be expressed in the rat p53 protein. In the present study, an immortalised, non-transformed liver epithelial cell line derived from a male F344 rat was transformed in vitro by aflatoxin B1 as demonstrated by tumour formation in nude mice. The transformation was dependent on metabolic activation of the aflatoxin B1. Transfection of DNA, extracted from these tumours, into NIH 3T3 fibroblasts conferred a stable, malignant transforming capacity. However, no mutations in codon 12 of the K-ras or codon 13 of the N-ras genes were detected in any of these tumours. These results indicate that in vitro transformation does not necessarily involve the same mutations, as those observed in vivo. Also, no mutations in codon 243 or adjacent codons of the p53 gene, paralleling those observed in the human cell line treated with aflatoxin B1, were detected. The results serve to emphasise the in vivo and in vitro variation in the oncogene activation in the same target organ or cell lines derived from that organ, even when using a single carcinogen activated by a known metabolic pathway.

Translocation of aged cyclodiene insecticide residues from soil into forage crops and pastures at various growth stages under field conditions.

Field trials were conducted to measure translocation of pesticides by summer and winter forage/pasture species from soil containing aged residues of heptachlor and, to a lesser extent, dieldrin. Substantial amounts of heptachlor epoxide, and lesser amounts of gamma-chlordane were translocated to plants from contaminated soil. Residue levels varied with crop species and stage of plant development. In summer crops residues were higher in soybean > cowpeas > lab-lab > Sorghum > millet > sweet saccaline at the grazing and mature stages. Compared to glasshouse studies undertaken previously, residues in crops grown under field conditions were much lower. This apparently reflects the lower soil moisture levels and the reduced rates of translocation. Heptachlor residues in winter crops were highest in Saia oats > Berseem clover > Haifa clover > Cassia oats > Tetila ryegrass > Schooner barley > Shaftal clover > Hunter river lucerne at the grazing stage. There were no detectable levels in barley and oats at the mature stage. No dieldrin residues were translocated into the various crop species.