Antimutagenic and mutagenic potentials of Chinese radish.

The edible part of fresh Chinese radish was chopped into small pieces, lyophilized, and then extracted sequentially with hexane, chloroform, and methanol. The solvent in each fraction was removed by evaporation under reduced pressure at 50-55 degrees C, and the residue was dissolved in dimethylsufoxide just before being tested for antimutagenicity as well as mutagenicity using the Salmonella/mammalian microsome mutagenicity test. We found that none of the three fractions exhibited any mutagenicity toward S. typhimurium strains TA98 and TA100 when tested either in the presence or absence of S-9 mix. Interestingly, however, hexane and chloroform extracts could strongly inhibit the mutagenicities of both direct mutagens (e.g., 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide and sodium azide) and indirect mutagens (e.g., aflatoxin B1). In contrast, however, these two fractions did not inhibit the mutagenicity of benzo[a]pyrene, which is also an indirect mutagen. Both hexane and chloroform extracts could also markedly inhibit the activities of rat liver aniline hydroxylase and aminopyrine demethylase. The methanol fraction could inhibit neither the mutagenicities of direct or indirect mutagens tested nor the activities of those two rat liver enzymes. Results of the present study demonstrate that Chinese radish may not contain any mutagenic compound but does contain some nonpolar compounds with antimutagenic activity toward both direct and indirect mutagens. In addition, the antimutagenic activity toward aflatoxin B1 may be partly due to the inhibition of enzymes necessary for activation of this mutagen.

Effect of varying the onset of exposure to DDT on its modulation of AFB1-induced hepatocarcinogenesis in the rat.

We have previously reported that p,p'-dichlorodiphenyl-trichloroethane (DDT) inhibited the hepatocarcinogenicity of aflatoxin B1 (AFB1) if given to rats 1 week prior to AFB1 but could enhance the carcinogenesis when given 1 week after the completion of AFB1 treatment. However, simultaneous administration of DDT with AFB1 resulted in a slight reduction in the incidence of liver tumours. In the present experiment in which the dose of AFB1 was reduced to about half of that used previously, we observed that DDT markedly inhibited the hepatocarcinogenesis if given to animals starting at the same time with AFB1. On the other hand, giving DDT to animals starting in the middle of AFB1 treatment resulted in a significant enhancement of hepatocarcinogenesis. DDT exhibited a maximal tumour promoting effect when given either 1 or 3 weeks after completion of AFB1 treatment. It enhanced the number of animals bearing liver carcinomas as well as the number of carcinomas per animal. Determination of gamma-glutamyltranspeptidase in the serum revealed that the activity increased only in animals bearing big and/or a number of carcinomas in the livers especially in those promoted by DDT. These results therefore demonstrated that DDT will act as an inhibitor of AFB1-induced hepatocarcinogenesis if it is given to animals starting either prior to or at the same time as carcinogen. On the other hand, it will act as a tumour promoter if given to animals starting either in the middle of or after the completion of AFB1 treatment.

Genotoxicity and cell proliferative activity of a nitrosated Oroxylum indicum Vent fraction in the pyloric mucosa of rat stomach.

In vivo genotoxic activity and cell proliferative activity were examined in the stomach mucosa of male F344 rats by in vivo short-term methods after oral administration of a nitrosated Oroxylum indicum Vent (OiV) fraction, which had been found to be mutagenic without S9 mix to Salmonella typhimurium TA98 and TA100. Administration of the nitrosated OiV fraction at doses of 1 and 2 g/kg body weight induced dose-dependent DNA single-strand scission (p less than 0.02), determined by the alkaline elution method, in the stomach pyloric mucosa 2 h after its administration: a dose of 2 g/kg body weight induced an 18-fold increase in the DNA elution rate constant. Administration of the nitrosated OiV fraction at doses of 0.7-2.8 g/kg body weight also induced dose-dependent increases, up to 11-fold (p less than 0.05), in replicative DNA synthesis in the stomach pyloric mucosa 16 h after its administration. Moreover administration of the nitrosated OiV fraction at doses of 0.25-2.0 g/kg body weight induced dose-dependent increases, up to 100-fold, in ornithine decarboxylase activity in the stomach pyloric mucosa with a maximum 4 h after its administration. These results demonstrate that the nitrosated OiV fraction has genotoxic and cell proliferative activity in the pyloric mucosa of rat stomach in vivo.

Quantitative measurements of the changes in protein thiols in cervical intraepithelial neoplasia and in carcinoma of the human uterine cervix provide evidence for the existence of a biochemical field effect.

Quantitative micromethods have been used for measuring reactive protein thiols (PSHr), total reactive protein sulfur (TRPS), total protein thiols (PSHt), and protein disulfides (PDS) in fixed frozen sections of human uterine cervix. PSHr and TRPS were stained using 2,2'-dihydroxy-6,6'-dinaphthyl disulfide; PSHt and PDS were stained using mercurochrome methods. Microspectrophotometric measurements were made on the stained sections using a microdensitometer with associated data processing; the results obtained for areas of epithelium and stroma were converted to absorbance values per micron 2. Samples of uterine cervix that were diagnosed as containing cervical intraepithelial neoplasia (CIN) I-III or carcinoma were examined and compared with samples of normal uterine cervix. Measurements were made not only on identified lesions but also on apparently normal tissue obtained from the same cervix. Epithelial/stroma ratios (E/S) were calculated for PSHr, TRPS, PSHt and PSHt + PDS; in addition, the double ratios of PSHr/TRPS and PSHt/PSHt + PDS were also calculated for E/S. The mean E/S values for PSHr and PSHt were significantly different for all types of lesion compared with control samples. The E/S ratios for apparently normal tissue obtained from cervices with CIN or carcinoma were also significantly different compared with corresponding control values, indicating a field effect. There was a considerable degree of overlap between individual values in the control groups versus those obtained with each type of lesion. The corresponding mean E/S values for TRPS and for PSHt + PDS in the samples containing lesions were not significantly different from control means except for the group containing CaCx. However, the mean values for the double ratios (PSHr/TRPS and PSHt/PSHt + PDS) were significantly different in the groups containing lesions compared with the controls. Moreover, apparently normal tissue obtained from cervices containing CIN or carcinoma had different mean values compared with the controls, confirming the existence of a field effect. The degree of overlap of individual values in the lesion groups compared with the control values was much less with double ratio values than previously noted for single ratio values. In consequence, the double ratio measurements clearly discriminated CIN I + II and CIN-III from controls. Our data show that CIN is associated with marked changes in tissue protein thiols and disulfides and that these differences extend to neighboring apparently normal tissue indicative of a field effect.

Histophotometry of protein thiols and disulphides in tissue samples from the human uterine cervix and the skin reveals a "field effect" as well as an "extended field effect" of malignant tumours.

Fresh frozen and fixed serial sections were stained with 2,2'-dihydroxy-6,6'-dinaphthyldisulfide (DDD) and Fast blue B for reactive protein thiols (PSHr) and total reactive protein sulfur (TRPS). The mean optical densities of PSHr and TRPS determined histophotometrically at a distinct part of a tissue were related to each other (PSHr: TRPS). If this quotient has been determined, e.g. for normal epithelium and the adjacent stroma, both quotients can be related to each other by a double quotient (Q PSHr: TRPS). With the aid of the double quotient highly significant differences could be found between tumours and normal tissue from patients without tumour of the human uterine cervix. Similar differences exist between normal skin from healthy patients and skin tumours. Q PSHr: TRPS revealed similar differences to exist between normal tissue of patients without tumour and apparently normal tissue in the neighbourhood of tumours of the uterine cervix and of skin ("field effect" of tumours). Histophotometric investigations on abdominal skin (and skin of breast) showed highly significant differences between normal skin of patients without tumour and patients with various kinds of tumours of the uterine cervix, ovaries, liver and breast ("extended field effect" of tumours).

Mutagenicity and antimutagenicity of Thai medicinal plants.

Crude extracts and partially purified as well as purified fractions were prepared from three Thai medicinal plants, namely, Acanthus ebracteatus Vahl, Plumbago indica Linn, and Rhinacanthus nasuthus Kurz, and then tested for their mutagenic and antimutagenic potentials using the Salmonella/microsome mutagenicity test. All fractions tested were not mutagenic toward either strain TA98 or TA100 whether tested in the presence or absence of S-9 mix. Interestingly, however, various fractions--especially those extracted by organic solvents such as petroleum ether, hexane, and chloroform, as well as some purified compounds from these plants--could strongly inhibit the mutagenicity of aflatoxin B1 (AFB1), an indirect mutagen, when tested in the presence of S-9 mix but not that of 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), which does not require metabolic activation for its mutagenicity. Furthermore, these fractions could markedly inhibit the activity of rat liver aniline hydroxylase, which is one of the cytochrome-P450-mediated reactions. These results therefore suggest that these Thai medicinal plants contain an antimutagen(s) which inhibits chemical mutagenesis by inhibiting the enzyme activities necessary for activation of indirect mutagens/carcinogens. Identification as well as anticarcinogenicity of purified compounds of these plants are being investigated in our laboratory.

Histophotometric quantification of the field effect and the extended field effect of tumors.

Histophotometric investigations have been made on samples of human skin. Fresh frozen serial sections were fixed and stained for either reactive protein thiols (PSHr) or total reactive protein sulphur (TRPS) using modifications of the DDD-Fast blue B-method. In addition, total protein thiols (PSHt) were stained with the Mercurochromcyanide-method, and proteins were stained using a modified amido-black procedure. Significant differences were found between the different tumours investigated and normal tissue, and also between apparently normal tissue adjacent to the tumours and normal tissue from patients without tumour. To reveal such tumour-related changes of apparently normal tissue, termed the field effect of tumours, a double quotient had to be calculated from the PSHr-and TRPS-values determined from both epithelium (epidermis) and connective tissue. In addition, abdominal skin was investigated from patients without tumour and patients with tumours of the female genital tract, liver or breast. With the aid of the double quotient procedure, highly significant differences were found between normal abdominal skin of patients without tumours versus similar samples taken from patients with tumours. The tumour-related changes found with abdominal skin distant from the tumours have been termed the extended field effect of tumours. These general tumour-related changes, independent of the size, state or degree of malignancy of the distant tumour, could be shown to be due to changes in abdominal dermis.

Failure of diterpenes from Jatropha curcas to induce mutation in Salmonella typhimurium TA98 and TA100.

No mutagenicity of five diterpenes isolated from the roots of Jatropha curcas was detected by Ame's test using both TA98 and TA100 tester strains with or without S-9 fraction from livers of rats treated with polychlorinated biphenyl.

Carcinogenicity of an oxidation product of p-phenylenediamine.

para-Phenylenediamine (p-PD), a widely used aromatic amine in the preparation of commercial oxidative-type hair dyes, has been previously demonstrated to have neither mutagenic activity to Salmonella typhimurium nor carcinogenic activity in rats and mice. In this study, the mutagenicity of p-PD after an oxidation by hydrogen peroxide towards S. typhimurium TA98 and its carcinogenicity in Wistar rats were examined both by topical application to the shaved skin and by s.c. injection. The oxidation product was found to be strongly mutagenic to the bacterial tester strain in the presence of rat liver S-9 fraction. Interestingly, in female rats, both topical application and s.c. injection for 18 months of oxidized p-PD could induce a statistically significant incidence of mammary gland tumors (greater than 50%, P less than 0.05). In addition, uterine tumors and soft tissue tumors of both malignant and benign types were also significantly induced (43% and 57%, P less than 0.05) in the s.c. injection group. On the other hand, tumors of mammary gland and soft tissue were not observed in male rats under similar experimental conditions. However, tumors of other organs including liver, kidney, adrenal gland, thyroid gland, urinary bladder and lung were occasionally observed in male rats of both groups and might be related to the p-PD treatment.

Protein thiols in normal and neoplastic human uterine cervix.

Protein-thiol groups that react with dihydroxydinaphthyl disulphide during a 7 h incubation (so-called reactive protein thiols, PSHr) have been quantitatively measured on sections of human uterine cervix by microcytospectrophotometry. Measurements were made on areas (1 micron 2) of epithelium and adjoining stroma in samples of normal cervix, and in samples obtained from patients with dysplasia, carcinoma-in-situ and invasive cancer. The ratio of PSHr in epithelium to stroma is substantially reduced in the pathological conditions compared with normal and in apparently normal adjacent areas. Such changes in PSHr are discussed in relation to the redox balance of the tissue, and free radical disturbances previously described.

The effect of vitamin A status on the differentiation and function of goblet cells in the rat intestine.

The prevalence of goblet cells in duodenal crypts decreased to 60% of normal at day 4 of deficiency. Villus and crypt length, total mucosal cell number, cell cycle time, and migration rate were unaffected at day 8 of deficiency. The carbohydrate content, protein content, and molecular size of high molecular weight glycoproteins (peak I) were unaffected at day 8 of deficiency. The incorporation of L-[3H]threonine (G) into proteins of all peaks were reduced to 60% of normal at day 8 of deficiency. The incorporation of L-[1-14C]fucose into glycoprotein fell to 25% of normal in peak I, and to 70 to 90% of normal in other peaks at day 8 of deficiency. In vitamin A deficiency, a defect seems to exist either in the rate of differentiation of intestinal goblet cells at a critical period, or in the differentiation of a subclass of vitamin A-sensitive goblet cells. Some possible explanations for the decreased synthetic rate of some glycoproteins in the presence of normal steady-state concentrations in A- rats are discussed.

Biochemical and immunological characterization and the synthesis of rat intestinal glycoproteins following the induction of rapid synchronous vitamin A deficiency.

Glycoproteins and proteins were extracted from segments or scrapings of the intestine in tube-fed, vitamin-A-deficient and control rats on the eight day after withdrawal of retinoic acid from the diet by using either 1% sodium dodecyl sulfate (SDS) or aqueous 5 mM EDTA (pH 7.4). They were then fractionated on columns of Sepharose 4B. Water-soluble peak I material contained large (Mr greater than 10(6); S20 = 11.7) glycoprotein aggregates which were rich in hexose, fucose and sialic acid. These aggregates dissociated into several non-identical glycoprotein and protein subunits upon treatment with dithiothreitol. The protein matrix was rich in threonine, valine, proline, serine, glutamate and aspartate. Peak II consisted of smaller proteins and glycoproteins, the latter with much lower carbohydrate content. Some peak II glycoproteins also dissociated into subunits in the presence of dithiothreitol. Peak III consisted mainly of a heterogeneous assortment of proteins, including some glycoproteins of low carbohydrate content. Antibodies either to peak Ii or to peak III reacted both with peaks II and III but not with peak I. The total weight, carbohydrate composition of glycoproteins and the ratio of carbohydrate to protein in the total extract or in each of the three fractions were not significantly affected in vitamin A deficiency despite decreased incorporation of all labeled precursors. Rather, the relatively lower incorporation (approx. 0.8) of radioactive sulfate, D-glucosamine and L-fucose into total SDS-soluble duodenal glycoproteins of vitamin-A-deficient rats could be explained on the basis of a reduced prevalence of goblet cells alone. In contrast, the relative incorporation rate of L-fucose into peak I, but not into peaks II and III, ranged from 0.25 to 0.45, less than expected on the basis of fewer goblet cells alone. The incorporation of radioactive threonine into all protein fractions was reduced to 60% of normal in vitamin A deficiency. Thus, the well established observation that intestinal tissue of vitamin-A-deficient rats synthesizes high molecular weight glycoproteins poorly might be due to several interacting factors: (1) a reduced prevalence of goblet cells, (2) a lower rate of protein synthesis, (3) a lack of retinyl phosphate for the conformation of mannosyl or other carbohydrate derivatives, and (4) secondary, and as yet undefined, cellular changes which preferentially reduce the rate of synthesis of high molecular weight fucose- and sialic-acid-enriched glycoproteins.

The prevalence, metabolism and migration of goblet cells in rat intestine following the induction of rapid, synchronous vitamin A deficiency.

Experiments were conducted to evaluate critically the role of vitamin A in the maintenance and functional integrity of mucus-secreting goblet cells in rat small intestine. Essentially synchronous vitamin A deficiency was induced by the withdrawal of retinoic acid from mature, stringently-deficient male rats reared by feeding vitamin A-depleted weanlings diets first supplemented with and then lacking in 2 micrograms retinoic acid per gram diet in repeating 18 day:10 day cycles. Secondary inanition was minimized by force-feeding both deficient and control animals twice daily. Whereas the prevalence of oligomucus cells was unchanged, the number of goblet cells per duodenal crypt gland decreased abruptly to 60% of control values starting 2 to 3 days after the withdrawal of retinoic acid and then stabilized. The responses of mucus-secreting cells to atropine and pilocarpine were identical in vitamin A deficient and control animals. As studied with [3H]thymidine, the rate of division of epithelial cells and the migration rate of columnar and goblet cells out of the crypt gland and along the villus were also unaffected by vitamin A deficiency. We conclude that two populations of goblet cells exist in the intestine--one relatively insensitive and the other sensitive to vitamin A status. In vitamin A deficiency, the rate of differentiation of sensitive goblet cells from oligomucus cells and other precursor cells seems to be blocked.