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The effect of the mycotoxin diacetoxyscirpenol (DS) on the upper alimentary tract was studied on account of the association between the consumption of food contaminated by Fusaria and esophageal cancer. Previously it had been shown that a single high dose of DS induced basal cell replication in esophagus and in squamous and glandular stomach. To assess the significance of this effect in relation to the levels of exposure likely to be encountered by man and agricultural animals, it was essential to examine the dose response relationship. Also, the long-term effect of repeated intubations of DS, and of chronic feeding of DS at 10 ppm in the diet, was studied. Intubation of progressively lower doses of DS produced a decreasing effect on replication in esophagus and stomach, but at 0.06 mg/kg replication in squamous and glandular stomach was still more than in the control animals. Intubation repeated weekly for 6-8 weeks produced no detectable change in esophagus or stomach in the surviving animals which were killed at 9 months. When DS was fed in the diet, there was marked hyperplasia in the squamous stomach of two of the four animals which survived for 9 months. These results suggest that DS per se is not carcinogenic for esophagus or for stomach, and that exposure to occasional high doses does not cause persisting abnormalities in replication. However, repeated exposure to high doses would cause repeated periods of hyperplasia, and chronic exposure in some animals could result in continuing hyperplasia. Any increase in replication is likely to promote cancer by increasing the vulnerability of the gastric and esophageal mucosa to carcinogens.

Newborn rats and adult animals in which liver cell replication had been induced by partial hepatectomy were injected with 5-bromodeoxyuridine (BUdR), and were kept without further treatment for the remainder of their life span. No evidence for carcinogenicity of BUdR was obtained, but a dose-dependent shortening of the life span was observed after administration to neonatal animals.

Dimethylnitrosamine given after partial hepatectomy reduces the synthesis of DNA, histone, and, to a lesser extent, of nonhistone proteins, the inhibition being general rather than for specific nuclear proteins. The extent of inhibition of macromolecular synthesis is greatest when the carcinogen is given a few hours after the operation. As a higher incidence of hepatocellular carcinoma is induced when dimethylnitrosamine is injected later, during the wave of DNA replication, it appears that inhibition of nuclear protein synthesis is not a relevant factor in carcinogenesis, unless it relates to loss of a specific non-histone protein present in small amounts. Analysis of sequentially extracted groups of non-histone proteins by 2D electrophoresis did not reveal any changes produced by treatment with dimethylnitrosamine. Animals fed a diet containing diethylnitrosamine showed an increased incorporation of amino acid into histone. Electrophoretic analysis of non-histone proteins revealed two reproducible effects of feeding the carcinogenic diet: relative to the bulk of nuclear non-histone protein, there was a reduction in the amount of a slightly basic 65000 mol. wt. polypeptide, and an increase in the level of a high molecular weight protein that was almost undetectable in material from normal rats. As these changes were not induced by partial hepatectomy of normal animals, it is possible that they are related to malignancy rather than to the associated increase in cell replication.

Consumption by man of cereals contaminated by high levels of Fusaria mycotoxins has caused alimentary toxic aleukia, while chronic consumption at lower levels of contamination has been implicated in esophageal cancer in China and South Africa. Dietary treatment of animals with extracts of Fusaria cultures or with the trichothecene diacetoxyscirpenol (DS) caused esophageal hyperplasia but not cancer. The explanation could be that esophageal cancer is initiated by other factors, possibly by nitrosamines, and that Fusaria mycotoxins act either as co-carcinogens or as promoters as a result of their ability to stimulate cell replication. The effect of DS on replication in esophagus was therefore studied. As squamous stomach has a very similar histological structure to esophagus, the effect of DS on stomach was studied also. A high dose of DS given by gavage was shown by the bromodeoxyuridine-antibody technique to increase DNA replication in the basal cells of the esophagus and of the squamous and glandular stomach. For stomach, this correlated with an increased incorporation of tritiated thymidine into DNA, and an increase in ornithine decarboxylase activity. These effects had returned to normal by 7 days. The increased replication was apparently not a result of cell damage and restorative hyperplasia. It is suggested that, as has been proposed recently for butylated hydroxyanisole, DS may enhance carcinogenesis when exposure is sufficient to stimulate cell replication. This contrasts with the non-threshold action of initiating carcinogens. For man, acute exposure to the critical dose of DS probably occurs only under exceptional circumstances, as during outbreaks of alimentary toxic aleukia. Prolonged exposure to lower dose levels is more likely to be relevant.

The high susceptibility of certain organs, for example rat brain, to induction of cancer by N-nitroso-N-alkyl-ureas, has been related to a low ability to remove O6-alkylguanine (O6AG) from DNA. It is therefore reasonable to ask why mouse brain, in which there is also a slow disappearance of O6AG from DNA after treatment with nitroso-alkyl-ureas, is not susceptible and why, in mice, thymus and lung are the main target organs. The explanation of the species difference could lie in the fact that replication of alkylated DNA is an essential event in initiation. If nitroso-alkyl-ureas had a greater inhibitory effect in some organs than in others, replication might be inhibited until after the O6AG had been removed, so preventing replication of DNA while still alkylated. This concept was tested by comparing the effect of N-nitroso-N-methyl-urea (NMU) on incorporation of [3H]TdR into DNA of relevant organs in Wistar rats and C57BL mice, and by determining ability to remove O6AG from DNA by measuring the alkyl acceptor protein (AAP) concentrations in these organs. No evidence was obtained that the AAP content was lower or inhibition of replication was less extensive in the organ of the species more susceptible to carcinogenesis than in the same organ of the less susceptible species.

A single injection of dimethylnitrosamine (DMN) given to adult rats induces hepatocellular carcinoma if given during the period of restorative hyperplasia following partial hepatectomy. The effect of DMN on the sequence of biochemical events in regenerating liver is therefore of interest. As carcinogenesis is likely to involve a change in control of gene expression, and as evidence suggests that non-histone chromosomal proteins (NHP) are involved in gene control, the effect of DMN on synthesis of NHP in regenerating liver was studied. A well-defined group of NHP, the high mobility group (HMG), known to be specifically associated with DNA active in transcription, were investigated. Partial hepatectomy was found to cause a large increase in incorporation of [3H]lysine into HMG proteins; this did not occur immediately, but was apparent at the time of DNA synthesis and of mitosis. DMN did not alter the relative amounts of different HMG proteins in relation to histone H1, but the carcinogen caused a considerable reduction in incorporation of [3H]lysine. This decreased synthesis of the proteins which may function as gene derepressors may well be relevant in carcinogenesis.

Epidemiological evidence from China and South Africa has implicated Fusaria mycotoxins in the etiology of esophageal cancer, although treatment of animals with extracts of Fusaria cultures did not cause cancer of the esophagus. Fusaria are the major producers of trichothecenes, and animal experiments have shown that these mycotoxins can damage the esophagus but they have not been shown to cause esophageal cancer. A plausible concept is therefore that esophageal cancer is initiated by the potent environmental esophageal carcinogens, certain nitrosamines, but that the levels of exposure are too low to cause clinical cancer unless their effects are enhanced by additional risk factors. Among the most likely enhancing factors in the regions mentioned above are Fusaria mycotoxins. As trichothecenes are known to inhibit sulphydryl-dependent reactions and to inhibit protein synthesis, experiments were carried out to determine whether potentiation of cancer could be mediated via inhibition of the DNA repair protein O6-methylguanine-DNA methyl transferase (O6MG-MT). The effect of diacetoxyscirpenol (DS) on O6MG-MT was studied. Chronic or acute treatment with DS did not alter the level of O6MG-MT in esophagus, or affect the depletion which occurs after injection of methylbenzylnitrosamine, or alter the rate of reappearance of O6MG-MT. A high dose of DS induced O6MG-MT in liver. These results suggest that if trichothecenes are risk factors for esophageal cancer, the effect is unlikely to be mediated by inhibition of O6MG-MT. Induction of the repair protein in liver may be relevant in the animal toxicoses caused by consumption of trichothecenes, but is unlikely to be implicated in the etiology of liver cancer in man.

3-Methyladenine is one of the major products formed by reaction of a large number of environmental methylating agents with DNA in vivo and in vitro. In spite of the rapid spontaneous depurination of this base an enzyme, 3-methyladenine DNA glycosylase, has been shown to catalyse its excision. The relevance of this enzyme in carcinogenesis induced by alkylating agents was studied. Acute or chronic treatment of rats with diethylnitrosamine or with N-acetylaminofluorene caused a slight increase in glycosylase activity in liver. Experiments with liver regenerating after partial hepatectomy showed a similar increase to occur at the time of DNA replication. It could be that the increase found after treatment with carcinogens was related to the accompanying increase in cell replication, rather than being the result of a specific induction by the carcinogen. Glycosylase activity was found to be higher in the liver of the rabbit and cat than in rat or hamster liver. Organ differences (liver, kidney and brain of the rabbit) were smaller than the species differences found for enzyme activity in liver.

The sequence of histological changes leading to oesophageal cancer in animals after treatment with various nitrosamines has been studied previously using systems which induced cancer relatively slowly. The more potent carcinogen, N-methyl-N-benzylnitrosamine (MBzN), has been employed to induce cancer in a very much shorter time in experiments designed to detect factors which may potentiate oesophageal cancer. MBzN injected twice weekly for 4 weeks induces papillomas by 4 weeks and carcinomas by 9 weeks. The histological events occurring during the 4-week treatment period have been studied. Initially there was an increase in mitosis. After the second injection, destruction of basal cells occurred, with their complete disappearance from stretches of the epithelium, accompanied by a massive influx of inflammatory cells, and disruption of the normal architecture of the oesophageal epithelium. After 1-2 weeks the numbers of inflammatory cells decreased, concurrently with basal cell hyperplasia and dysplasia, where replicating basal cells penetrated down into the submucosa and upwards towards the lumen. Thickening of the spinous and granular layers of the epithelium and hyperkeratosis were evident, and after 30 days a macroscopically visible papilloma was present. It therefore appears that resistance develops to the toxic inhibitory effect of the carcinogen, and this allows the abnormal replication of basal cells to occur even in the continued presence of the carcinogen. The biochemical processes underlying this development of resistance are being studied, and the effects of possible potentiating factors on the sequence of events occurring during carcinogenesis are being used to test for dietary factors which may enhance oesophageal cancer.

Summary Evidence suggests that replication of damaged DNA is an essential early event in carcinogenesis, i.e. replication must take place before the DNA damage caused by the carcinogen has been repaired. When cancer is induced by feeding a diet containing diethylnitrosamine (DEN), a critical minimum period of feeding is essential for induction of tumors within 9 months. This critical time may be the period necessary for stimulation of de novo replication or for inhibition of repair replication. The concept was tested by studying de novo and repair replication during administration of DEN, using a technique for fractionation of nuclei into replication and non-replicating diploids and tetraploids. [3H]- Thymidine ([3H]TdR) incorporation in vivo into replicating nuclei represented de novo replication, while hydroxyurea (HU)-resistant incorporation into non-replicating nuclei represented repair replication. De novo replication increased to a maximum after approx. 6 weeks feeding, while at approximately the same time repair replication was apparently unaltered, as tested by injection of a high dose of dimethylnitrosamine (DMN). The results therefore support the idea that the critical minimum time of feeding DEN is necessary for the development of restorative hyperplasia, when the increased rate of DNA replication increases the probability of synthesis of DNA taking place before damage caused by reaction with the carcinogen has been repaired.

To study the roles of DNA polymerases alpha and beta during replication and repair of damaged DNA, use was made of the fact that during chronic treatment with carcinogens, replication and repair do not necessarily follow the same time sequence. Early cell damage and restorative hyperplasia cause a transient wave of DNA synthesis, while repair replication might be expected to continue throughout the period of treatment with the carcinogen. N-acetylaminofluorene (AAF) was fed in the diet for periods of up to 35 weeks, and at intervals during the feeding period measurements were made of DNA synthesis in vivo, and of DNA polymerases alpha and beta as assayed in vitro after fractionation. The activity of polymerase alpha increased and decreased with the transient early wave of DNA synthesis. Polymerase beta showed an initial rapid increase in activity which peaked before the increase in DNA synthesis, and then decreased. The decrease in activity may be due to the fact that, although AAF continues to be fed in the diet, the foci and nodules which develop no longer metabolise the carcinogen to a form which damages DNA. Thus replication occurs in the nodules while DNA damage and repair occur in the surrounding non-neoplastic liver. With the rapid growth of nodules there is overall an increase in neoplastic tissue, a relative decrease in nonneoplastic tissue, and thus a relative decrease in DNA damage, repair, and induction of polymerase beta. Histological examination showed that by 35 weeks the conversion to neoplasia was virtually complete. These results support the concept that polymerase alpha functions in de novo replication of DNA, and is induced during cell replication, while polymerase beta functions in repair replication, and increases in activity during chronic damage to DNA. Whether it is induced by treatment with carcinogens depends on the duration of treatment, and on other processes (e.g. metabolism of the carcinogen) which take place during the development of malignancy.

It has often been suggested that the high molecular weight DNA polymerase alpha of eukaryotes plays a role in de novo replication of DNA, while the low molecular weight polymerase beta is involved in repair replication. Previous studies have shown that when diethylnitrosamine is fed in the diet to rats it causes after a few weeks an increase in de novo replication of DNA, which then returns to normal values. In contrast, repair replication may be expected to continue throughout the feeding period. Study of DNA polymerase activity in livers of animals during carcinogenesis showed that an increase in polymerase alpha occurred at the time of increased de novo replication, while there was a gradual increase in polymerase beta during the time diethylnitrosamine was present in the diet. When diethylnitrosamine treatment was stopped, there was a rapid drop in polymerase beta activity. These results support the view that the polymerase alpha is involved in DNA replication, that the polymerase beta functions in repair replication, and that the beta enzyme can be induced by chronic damage to DNA.

The fact that the only chemicals known to be potent carcinogens for the esophagus in animals are certain nitrosamines suggests that these environmental carcinogens could be a cause of human esophageal cancer. Epidemiological investigations support this concept. The level of exposure alone is not considered sufficient to account for the very high incidence of the disease in certain regions, but potentiating factors have been shown to have a dramatic effect on nitrosamine-induced esophageal cancer in animal experiments. A likely enhancing factor is consumption of food contaminated by molds, especially by Fusaria spp, a group known to produce trichothecene mycotoxins. The effect of simultaneous treatment with diacetoxyscirpenol (DS) on methyl-benzyl-nitrosamine (NMBzA)-induced esophageal cancer was studied. Feeding a diet containing DS at 10 ppm for 10 weeks caused thickening of the basal cell layer of the esophageal epithelium, but feeding DS (10 ppm) simultaneously with NMBzA (4, 8, 16 ppm) for 10 weeks, or feeding a lower dose of DS with NMBzA for a longer period, or administration of DS per os at intervals during NMBzA treatment, did not potentiate but possibly reduced esophageal tumors. Toxicity, revealed by reduced growth rate of DS-fed animals, may have inhibited carcinogenesis. In contrast to the rapid potentiating effect of zinc deficiency, DS does not appear to cause an early enhancement of esophageal cancer.