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3165. Azoxymethane and colon carcinogenesis
Toxicology,Cancer research
262
releaseby a proteolytic enzyme is the result of metabolic changes in the mast cells. The third paper reports that of four food technologists who were occupationally exposed to high concentrations of papain dust in air, two developed an acute asthmatic reaction followed by signs of obstructive airways disease that persisted as long as they remained exposed to papain. When all the subjects were tested 17 months after exposure, their ventilatory capacity was within normal limits. No evidence of emphysema was sought. Two of the subjects, one with and one without a history of allergy, showed a heightened bronchial reactivity when challenged with methacholine. In view of the emphysema that has been noted in experimental animals exposed to papain by inhalation, the minimal effects on respiratory function hitherto observed in people encountering high concentrations of papain dust seem remarkable. 3163. Negative Ames test for optical brighteners
McGregor, D. B. & Ainsworth, L. (1976). Lack of mutagenic activity in Salmonella typhimurium of four optical brighteners. Mutation Res. 40, 169. The reported induction of mutations in yeast by optical brighteners (Gillberg & Aman, Mutation Res.
1971,13, 149)was not confirmed in some later experiments (Kilbey & Zetterberg, ibid 1973, 21, 73). Further support for the negative findings has now been obtained using the Ames test. The strains of Salmonella typhimurium used were TA1535 and TAl538, the former for detecting base-pair substitutions and the latter for frameshift mutagens. Tests for the reversion of histidine-dependence in these two strains were carried out in the presenceand absenceof a rat-liver postmitochondrial supernatant as metabolic activator, and the optical brighteners tested were disodium 4,4’-bis-([4-anilino-6-morpholino-1,3,5-triazin-2-yl]aino)stilbene-2,2’-disulphonate, sodium 2-(4styryl-2-sulphophenyl)-2H-naphtho[1,2-dtriazole, disodium 4,4’-bis-([4-anilino-6-(Nmethyl-N-2-hydroxyethylamino)-1,3,5-triazin-2-y1]amino)stilbene-2,2’-disulphonate and disodium 4,4’bis-(2-sulphostyryl)biphenyl. Neither before nor after metabolic activation did any of the test substances show mutagenic activity to either bacterial strain, even at a concentration of 2 mg/plate. The possibility that penetration of the test materials into the bacteria was low is considered unlikely, since the cell-wall perrheability in these strains is relatively high; moreover, penetration of these compounds into yeast cells has been demonstrated although the cell walls of yeasts are generally less permeable to foreign compounds than are those of bacteria.
TOXICOLOGY 3164. The role of the got flora in toxicity
Reddy, B. G., Pohl, L. R. & Krishna, G. (1976). The requirement of the gut flora in nitrobenzene-induced methemoglobinemia in rats. Biochem. Pharmac. 25, 1119. The gut flora plays a major role in the reduction of p-nitrobenzoic acid in the rat, even when the compound is given parenterally (Cited in F.C.T. 1975, 13, 485), as was demonstrated in a study in which the nitro-reductase activity of the gut contents was virtually eliminated by antibiotic pretreatment. Many other nitro compounds are also reduced by the gut flora, often to metabolites that are more toxic than the parent compound (ibid 1970, 8, 120). The relationship between the reduction of nitrobenzene by the gut flora and methaemoglobin formation in uiuo has now been investigated. When nitrobenzene (2OOmg/kg body weight in sesameoil) was injected ip into normal (control) rats,
CANCER 3165. Azoxymethane
and colon carcinogenesis
Narisawa, T., Wang, C.-Q. & Weisburger, J. H. (1976). Azoxymethane-induced liver hemangiosarcomas in inbred strain-2 guinea pigs. J. natn. Cancer Inst. 56, 653.
about 3040% of the blood haemoglobin was converted to methaemoglobin within l-2 hr. The same dose given to germ-free or antibiotic-pretreated rats produced no measurable methaemoglobinaemia even after 7 hr, but in initially germ-free animals acclimatized in a normal animal room for 7 days it induced methaemoglobinaemia to the same extent as in control rats. In liver, kidney and gut-wall homogenates, the rate of aniline formation from nitrobenzene did not differ significantly whether the tissues were taken from normal germ-free or acclimatized germ-free rats, being low in all cases(about 2-3 m-no1 aniline/mg protein/hr in the liver and gut wall and 05-08 nmol/mg protein/hr in the kidney). However, the gut contents from control and acclimatized germfree rats produced aniline at a much faster rate (1I-15 nmol/mg protein/hr), whereas in actual germfree rats the nitro-reductase activity of the gut contents was virtually zero. It appears, therefore, that the gut flora present in normal and acclimatized animals was responsible for nitrobenzene reduction and subsequent methaemoglobin formation.
RESEARCH
Azoxymethane (AOM), on intrarectal instillation, produced a higher incidence of colonic tumours in germ-free than in conventional rats, indicating that metabolism by the gut flora was not a necessaryprerequisite for carcinogenesisat this site (Cited in F.C.T 1976,14,79). Mice and hamsters, but not guinea-pigs,
Cancer research were also shown to be susceptible to AOM-induced cancer of the colon (Weisburger, Dis. Colon Rectum 1973, 16, 431). Tumours at this site have, however, developed in guinea-pigs after intrarectal instillation of N-methyl-N-nitrosourea (Narisawa et al. J. mtn. Cancer Inst. 1975, 54, 785), and a further attempt to induce colon cancer in this species with AOM has now been made. Female guinea-pigs were instilled intrarectally with 1 mg AOM as a 0.2% aqueous solution twice weekly for 33 wk, and were subsequently maintained until death which occurred between wk 32 and 54. Treatment was temporarily discontinued in some animals which showed loss of weight and appetite and general weaknessafter 16 wk, and the total dose administered thus ranged from 52 to 64mg. Autopsy revealed that 15 of the 16 guinea-pigs had developed multiple liver haemangiosarcomas, which had invaded and destroyed the surrounding liver tissue and which, in three animals, had metastasized to the lungs, spleen,
263
adrenal glands and kidneys. In addition there was usually evidence of hepatocellular injury, fatty degeneration of the liver cells, post-necrotic fibrosis and bile-duct hyperplasia. No neoplasms were found in other organs, and preneoplastic changes were not observed microscopically in random sections of the large intestine. As AOM requires metabolic activation to be carcinogenic (Weisburger, lot. cit.), the findings suggest’ that the colonic mucosa of the guinea-pig, unlike that of the rat, does not contain sufficient amounts of the required enzymes. On the other hand, AOM must be metabolized to an active form by specific cells in the guinea-pig liver. The high incidence of haemangiosarcomas in this study was unexpected, because such tumours have been reported previously only in rats given AOM together with an immunosuppressive treatment, while rats treated (SC)with AOM alone developed only colon cancer (Kroes et al. Cancer Res. 1975, 35, 2651).
262
releaseby a proteolytic enzyme is the result of metabolic changes in the mast cells. The third paper reports that of four food technologists who were occupationally exposed to high concentrations of papain dust in air, two developed an acute asthmatic reaction followed by signs of obstructive airways disease that persisted as long as they remained exposed to papain. When all the subjects were tested 17 months after exposure, their ventilatory capacity was within normal limits. No evidence of emphysema was sought. Two of the subjects, one with and one without a history of allergy, showed a heightened bronchial reactivity when challenged with methacholine. In view of the emphysema that has been noted in experimental animals exposed to papain by inhalation, the minimal effects on respiratory function hitherto observed in people encountering high concentrations of papain dust seem remarkable. 3163. Negative Ames test for optical brighteners
McGregor, D. B. & Ainsworth, L. (1976). Lack of mutagenic activity in Salmonella typhimurium of four optical brighteners. Mutation Res. 40, 169. The reported induction of mutations in yeast by optical brighteners (Gillberg & Aman, Mutation Res.
1971,13, 149)was not confirmed in some later experiments (Kilbey & Zetterberg, ibid 1973, 21, 73). Further support for the negative findings has now been obtained using the Ames test. The strains of Salmonella typhimurium used were TA1535 and TAl538, the former for detecting base-pair substitutions and the latter for frameshift mutagens. Tests for the reversion of histidine-dependence in these two strains were carried out in the presenceand absenceof a rat-liver postmitochondrial supernatant as metabolic activator, and the optical brighteners tested were disodium 4,4’-bis-([4-anilino-6-morpholino-1,3,5-triazin-2-yl]aino)stilbene-2,2’-disulphonate, sodium 2-(4styryl-2-sulphophenyl)-2H-naphtho[1,2-dtriazole, disodium 4,4’-bis-([4-anilino-6-(Nmethyl-N-2-hydroxyethylamino)-1,3,5-triazin-2-y1]amino)stilbene-2,2’-disulphonate and disodium 4,4’bis-(2-sulphostyryl)biphenyl. Neither before nor after metabolic activation did any of the test substances show mutagenic activity to either bacterial strain, even at a concentration of 2 mg/plate. The possibility that penetration of the test materials into the bacteria was low is considered unlikely, since the cell-wall perrheability in these strains is relatively high; moreover, penetration of these compounds into yeast cells has been demonstrated although the cell walls of yeasts are generally less permeable to foreign compounds than are those of bacteria.
TOXICOLOGY 3164. The role of the got flora in toxicity
Reddy, B. G., Pohl, L. R. & Krishna, G. (1976). The requirement of the gut flora in nitrobenzene-induced methemoglobinemia in rats. Biochem. Pharmac. 25, 1119. The gut flora plays a major role in the reduction of p-nitrobenzoic acid in the rat, even when the compound is given parenterally (Cited in F.C.T. 1975, 13, 485), as was demonstrated in a study in which the nitro-reductase activity of the gut contents was virtually eliminated by antibiotic pretreatment. Many other nitro compounds are also reduced by the gut flora, often to metabolites that are more toxic than the parent compound (ibid 1970, 8, 120). The relationship between the reduction of nitrobenzene by the gut flora and methaemoglobin formation in uiuo has now been investigated. When nitrobenzene (2OOmg/kg body weight in sesameoil) was injected ip into normal (control) rats,
CANCER 3165. Azoxymethane
and colon carcinogenesis
Narisawa, T., Wang, C.-Q. & Weisburger, J. H. (1976). Azoxymethane-induced liver hemangiosarcomas in inbred strain-2 guinea pigs. J. natn. Cancer Inst. 56, 653.
about 3040% of the blood haemoglobin was converted to methaemoglobin within l-2 hr. The same dose given to germ-free or antibiotic-pretreated rats produced no measurable methaemoglobinaemia even after 7 hr, but in initially germ-free animals acclimatized in a normal animal room for 7 days it induced methaemoglobinaemia to the same extent as in control rats. In liver, kidney and gut-wall homogenates, the rate of aniline formation from nitrobenzene did not differ significantly whether the tissues were taken from normal germ-free or acclimatized germ-free rats, being low in all cases(about 2-3 m-no1 aniline/mg protein/hr in the liver and gut wall and 05-08 nmol/mg protein/hr in the kidney). However, the gut contents from control and acclimatized germfree rats produced aniline at a much faster rate (1I-15 nmol/mg protein/hr), whereas in actual germfree rats the nitro-reductase activity of the gut contents was virtually zero. It appears, therefore, that the gut flora present in normal and acclimatized animals was responsible for nitrobenzene reduction and subsequent methaemoglobin formation.
RESEARCH
Azoxymethane (AOM), on intrarectal instillation, produced a higher incidence of colonic tumours in germ-free than in conventional rats, indicating that metabolism by the gut flora was not a necessaryprerequisite for carcinogenesisat this site (Cited in F.C.T 1976,14,79). Mice and hamsters, but not guinea-pigs,
Cancer research were also shown to be susceptible to AOM-induced cancer of the colon (Weisburger, Dis. Colon Rectum 1973, 16, 431). Tumours at this site have, however, developed in guinea-pigs after intrarectal instillation of N-methyl-N-nitrosourea (Narisawa et al. J. mtn. Cancer Inst. 1975, 54, 785), and a further attempt to induce colon cancer in this species with AOM has now been made. Female guinea-pigs were instilled intrarectally with 1 mg AOM as a 0.2% aqueous solution twice weekly for 33 wk, and were subsequently maintained until death which occurred between wk 32 and 54. Treatment was temporarily discontinued in some animals which showed loss of weight and appetite and general weaknessafter 16 wk, and the total dose administered thus ranged from 52 to 64mg. Autopsy revealed that 15 of the 16 guinea-pigs had developed multiple liver haemangiosarcomas, which had invaded and destroyed the surrounding liver tissue and which, in three animals, had metastasized to the lungs, spleen,
263
adrenal glands and kidneys. In addition there was usually evidence of hepatocellular injury, fatty degeneration of the liver cells, post-necrotic fibrosis and bile-duct hyperplasia. No neoplasms were found in other organs, and preneoplastic changes were not observed microscopically in random sections of the large intestine. As AOM requires metabolic activation to be carcinogenic (Weisburger, lot. cit.), the findings suggest’ that the colonic mucosa of the guinea-pig, unlike that of the rat, does not contain sufficient amounts of the required enzymes. On the other hand, AOM must be metabolized to an active form by specific cells in the guinea-pig liver. The high incidence of haemangiosarcomas in this study was unexpected, because such tumours have been reported previously only in rats given AOM together with an immunosuppressive treatment, while rats treated (SC)with AOM alone developed only colon cancer (Kroes et al. Cancer Res. 1975, 35, 2651).