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Genotoxicity and cell proliferative activity of a nitrosated Oroxylum indicum Vent fraction in the pyloric mucosa of rat stomach
Mutation Research, 281 (1992) 55-61
55
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
MUTLET 00609
Genotoxicity and cell proliferative activity of a nitrosated Oroxylum indicum Vent fraction in the pyloric mucosa of rat stomach Anong Tepsuwan
1 Chie Furihata 2, Wannee Rojanapo 1 and Taijiro Matsushima 2
1 Biochemistry and Chemical Carcinogenesis Section, Research Division, National Cancer Institute, Bangkok (Thailand) and 2 Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Tokyo 108 (Japan)
(Received 15 February 1991) (Revision received 12 September 1991) (Accepted 13 September 1991) Keywords: Oroxylum indicum Vent; DNA single-strand scission; Ornithine decarboxylase;Replicative DNA synthesis; Rat stomach
mucosa
Summary 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 $9 mix to Salmonella typhimurium TA98 and TA100. Administration of the nitrosated OiV fraction at doses of 1 and 2 g / k g body weight induced dose-dependent D N A single-strand scission ( p < 0.02), determined by the alkaline elution method, in the stomach pyloric mucosa 2 h after its administration: a dose of 2 g / k g body weight induced an 18-fold increase in the D N A elution rate constant. Administration of the nitrosated OiV fraction at doses of 0.7-2.8 g / k g body weight also induced dose-dependent increases, up to ll-fold ( p < 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 / k g 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.
Oroxylum indicum Vent (OiV, commonly called Pay-gaa in Thailand) belongs to the family Bignoniaceae. It is a traditional medicinal plant in Thailand used in the treatment of various kinds of diseases, such as stomach disorders, diarrhea, and dysentery, as a diaphoretic (Burkill,
Correspondence: Dr. C. Furihata, Department of Molecular Oncology, Institute of Medical Science, Universityof Tokyo, Shirokanedai, Minato-ku, Tokyo 108 (Japan).
1935), and as a styptic and antipyretic for the treatment of rheumatic swellings (Perry and Metzger, 1980). It contains oroxylin A (5,7-dihydroxy-6-methoxyflavone), baicalein (5,6,7-trihydroxyflavone), chrysin and 6-methylbaialein (Shah et al., 1936; Bose and Bhattacharya, 1938; Subramanian and Nair, 1972a, b). The biological activities of this plant have been studied and results have shown that it has anti-inflammatory, antiallergic (Galikov and Brekhman, 1967) and antispasmodic (Dhar et al., 1968) activities. Crude ethanol and hexane extracts of this plant have no
56
mutagenic activity on Salmonella typhimurium TA98 or TA100 in either the presence or the absence of $9 mix. However, after nitrosation with nitrite under acidic conditions, these extracts become strongly mutagenic to Salmonella typhimurium strains TA98 and TA100 in the absence of $9 mix (Ieamworapong et al., 1989). These results suggest that a nitrosation product(s) in extracts of this plant is a direct-acting mutagen(s). A direct-acting mutagen/carcinogen produced by nitrosation in the acidic conditions of the stomach is thought to be a possible cause of human gastric cancer (Mirvish, 1983). Both tumor initiation and tumor promotion are thought to be important in human carcinogenesis. Therefore, the tumor-initiating and -promoting activities of the nitrosation product of OiV were examined to evaluate its potential hazard to humans. Furihata et al. (1984a, b, 1985a, 1989a) have developed in vivo short-term methods for evaluating possible tumor-initiating activity (genotoxicity) assessed as unscheduled DNA synthesis (UDS) and DNA single-strand scission determined by an alkaline elution method as markers, and also possible tumor-promoting activity (cell proliferative activity) using replicative DNA synthesis (RDS) and ornithine decarboxylase (ODC) as markers. In this study, we examined the possible tumor-initiating and -promoting activities of a nitrosated product(s) of OiV in the pyloric mucosa of rat stomach by these techniques after oral administration of an OiV fraction. The results indicate that the nitrosated product may have tumor-initiating and -promoting activities in the pyloric mucosa of rat stomach. Materials and methods
Preparation of nitrosated OiV fraction Dry bark of Oroxylum indicum Vent, purchased from a medicinal plant store in Bangkok, was chopped into small pieces and then blended to a powder. A crude ethanol extract was prepared by soaking the bark powder in 70% ethanol overnight and then filtering the mixture through Whatman No. 1 filter paper. The extraction process was repeated and the 2 filtrates were combined and evaporated to dryness at 55°C under
1000
800 c-
oo
600
o
% •-r
400
o
E
200
Z
0
I
0 20
I
I
I
50
100
200
Amount of OiV (IJg/plate) Fig. l. Appearance of mutagenicity to Salmonella typhimurium TA100 without $9 mix on treatment of OiV with nitrite. Nitrite concentrations 0 (X), 10 (o), 25 (zx), 100 (11), 250 ( o ) and 500 ( • ) raM.
reduced pressure in a rotary evaporator. The nitrosated product was prepared by a slight modification of the method of Takeda and Kanaya (1982). We tested nitrite concentrations of 10-500 mM (Fig. 1), and on the basis of the results we used 500 mM nitrite to prepare the nitrosated OiV fraction. Briefly, the ethanol extract was incubated at 37°C in the dark with 500 mM sodium nitrite in solution adjusted to pH 3 by addition of HC1. After incubation for 3 h, the mixture was centrifuged at 6000 × g for 15 min and the supernatant and the precipitate were separated. The supernatant was passed through a Sep-Pak C-18 cartridge to remove sodium chloride and sodium nitrite and also to concentrate the nitrosation product. The product was eluted from the cartridge with methanol and evaporated to dryness at 55°C in a rotary evaporator under reduced pressure. The precipitate that appeared after centrifugation was washed with cold distilled water and was dried in vacuo. The 2 fractions were combined and were used as the nitrosated OiV fraction. The mutagenicity of the samples was tested by the method of Maron and Ames (1983).
57
Animals and treatment Male Fischer rats ( F 3 4 4 / D u Crj: Charles River Japan, Inc., Kanagawa) 7-8 weeks old were given a limited amount of diet (4 g of commercial pellet diet (Nihon Clea, Tokyo) per rat of 200 g body weight) overnight to reduce their dietary stomach contents. The following day, they were given the nitrosated OiV fraction suspended in distilled, deionized water by gastric intubation. Control animals were given distilled, deionized water only. The stomach was removed at an appropriate time, opened along the greater curvature, and washed with sterile saline. The pyloric mucosa was scraped off with a razor blade or cut into small pieces with scissors and used for assays of DNA single-strand scission, RDS and ODC activity.
stomach was determined in in vitro organ culture in the presence of tritiated thymidine ([3H]dThd, 2.96 T B q / m m o l e , ICN Radiochemicals, Irvine, CA) as described previously (Furihata et al., 1984b, 1985a). The DNA fraction was extracted from the tissue and an aliquot was dissolved in ASC I1 (Amersham, Arlington Heights, IL). The incorporation of [3H]dThd into DNA was determined in a Packard Tricab 1500 liquid scintillation counter. The DNA contents of the DNA fractions were determined with 3,5-diaminobenzoic acid, with calf thymus DNA (Worthington Biochemicals Co., Freehold, N J) as a standard. Values for 5 individual rats were determind in each experiment. The results were analyzed statistically by Welch's test.
Alkaline elution of DNA
Ornithine decarboxylase (ODC) assay
A modification (Furihata et al., 1989a) of the method of Sina et al. (1983) was used for alkaline elution of DNA. The pyloric mucosa was obtained from rats 2 h after administration of the OiV fraction and lysed on a filter (Fluorinert, 1.0 /xm pore size and 25 mm diameter, Japan Millipore Ltd., Tokyo, Japan) with 3.0 ml of lysis solution (100 mM glycine, 25 mM E D T A and 2% sodium dodecylsulfate, pH 9.7) in the presence of proteinase K (1 m g / m l , Sigma Chemical Co., St. Louis, MO) for 60 min in the dark. After the lysis solution had passed through the filter, the filter was washed with 3 ml of a 2 mM E D T A solution, pH 10. Then DNA was eluted in the dark at 20-25°C with 30 ml of alkaline solution (20 mM E D T A and 1.95% tetra-n-propylammonium hydroxide, pH 12.1) at a flow rate of 0.05 ml/min. Fractions of 3.0 ml were collected and the DNA contents of the fractions of eluate and on the filters were determined with 3,5-diaminobenzoic acid (Tokyo Kasei Kogyo Co., Tokyo) (Kissane and Robins, 1958). The elution rate constant (ml-1) of D N A was calculated from a semi-logarithmic plot of the amount of DNA remaining on the filter (log scale) against the fraction number from the first to the fifth fraction. The results were analyzed statistically by Welch's test.
The mucosa of rat stomach was homogenized in ODC extraction buffer and ODC activity in the extract was determined using L-[1-~4C]ornithine (2.01 G B q / m m o l e , New England Nuclear, Boston, MA) as a substrate as described previously (Furihata et al., 1985a). Briefly, samples of pyloric mucosa from 3 rats were pooled and homogenized in 50 mM sodium phosphate buffer, pH 7.2, containing 0.1 mM pyridoxal phosphate and 0.1 mM ethylenediaminetetraacetic acid (EDTA). The homogenate was centrifuged at 16,000 rpm for 20 min at 4°C, and the supernatant was used for measurement of ODC activity. The protein contents of the extracts were determined by microassay as described by Heil and Zilling (1970) with bovine serum albumin as a standard. Results are shown as means for duplicate assays on pooled materials from 3 rats.
Repficative DNA synthesis (RDS) After administration of the nitrosated OiV fraction in vivo, RDS in the pyloric mucosa of the
Results
Induction of DNA single-strand scission Fig. 2 shows typical elution patterns of DNA obtained from the pyloric mucosa of control rats and those treated with the nitrosated OiV fraction. Fig. 3 shows the dose-dependent increase in the elution rate constant 2 h after administration of doses of 1 and 2 g / k g body weight. A dose of 2 g / k g body weight resulted in an 18-fold increase ( p < 0.02) in the elution rate constant. These results demonstrated that the nitrosated OiV
58
1.0
l
6000=
/..
3oooI ~{
g ==0.1
7< u_ ~ 0.05
Fracteon No
'
'0
I /
/':
"
o ,
Fig. 2. Typical alkaline elution patterns of D N A from the stomach pyloric mucosa of rats 2 h after treatment with the nitrosated OiV fraction at doses of 0 (HzO) (©), 1 ( A ) and 2 (•) g / k g body weight.
fraction caused significant induction of DNA single-strand scission in the pyloric mucosa of rat stomach after its oral administration at doses of 1 and 2 g / k g body weight.
0
0.7
1.4
2.8
Dose of nitrosated OiV fraction (g/kg body weight) Fig. 4. Dose-dependent increase in RDS in the pyloric mucosa 16 h after administration of the nitrosated OiV fraction. Values at each dose are for 5 individual rats. Points represent values for individual rats and bars show m e a n s of these individual values. Values were significantly different from control at doses of 1.4 g ( p < 0.02) and 2.8 g ( p < 0.05)/kg body weight by Welch's test.
g~ o
i 40
•
30 1
•
F
Stimulation of replicative DNA synthesis The nitrosated OiV fraction induced a dosedependent increase in RDS in the pyloric mucosa 16 h after its administration at doses of 0.7-2.8 g / k g body weight (Fig. 4). A dose of 2.8 g / k g body weight induced an ll-fold increase in RDS ( p < 0.05).
"
Induction of ornithine decarboxylase activity
Olqll
0
-
~
I
1
2
Dose of nitrosated OiV fraction (g/kg body weight) Fig. 3. Dose-dependent increase in elution rate constant of D N A from stomach pyloric mucosa of rats 2 h after administration of the nitrosated OiV fraction. Values at 1.0 and 2.0 g / k g body weight are significantly different from control by Welch's test ( p < 0.02).
Fig. 5 shows the induction of ODC activity in the pyloric mucosa of rat stomach with a maximum 4 h after administration of the nitrosated OiV fraction at a dose of 2 g / k g body weight. This maximum ODC activity, releasing 564 pmole C O z / 3 0 m i n / m g protein, was about 100-fold the control activity. A dose-dependent increase in ODC activity was induced in the pyloric mucosa 4 h after administration of the nitrosated OiV fraction at doses of 0.25-2.0 g / k g body weight (Table 1). We also observed a slight increase in ODC activity in the fundic mucosa with similar dose dependence as the activity in the pyloric mucosa. However, no
59 600 500 £ 400 E 300 cE 0 ~ 200 ,D
o 100 o c3 E O 0
I
I
I
4
8
16
I
24h
Time after administration Fig. 5. Induction of ODC activity in the pyloric mucosa of rat stomach by the nitrosated OiV fraction (2 g/kg body weight). Results are means for duplicate assays on pooled materials from the rats.
ODC induction was observed in the forestomach epithelium in a parallel experiment to that for the results in Table 1 (data not shown). These results suggest differences in the ability of the nitrosated OiV fraction to induce ODC activity in the pyloric mucosa, fundic mucosa and forestomach epithelium of rat stomach.
Stimulation of replicative DNA synthesis and induction of ornithine decarboxylase by non-nitrosated OiV Non-nitrosated OiV itself stimulated replicative DNA synthesis in the pyloric mucosa of rat
TABLE 1 DOSE-DEPENDENT INDUCTION OF ODC ACTIVITY IN THE PYLORIC MUCOSA OF RAT STOMACH BY THE NITROSATED OiV FRACTION Dose (g/kg body weight)
ODC activity (pmole CO 2/30 min/mg protein)
0 0.25 0.50 1.0 2.0
6.3 16.2 34.5 75.6 174.1
stomach to some extent. Replicative DNA synthesis in the untreated control group (4 rats) was 163 _+25 dpm [3H]dThd//xg DNA. Doses of 1.0 and 2.0 g / k g body weight of non-nitrosated OiV (4 rats in each group) induced replicative DNA synthesis of 307 _+42 (p < 0.001) and 365 _+ 124 (p < 0.05) dpm [3H]dThd//xg DNA, respectively. Thus the nitrosated OiV fraction caused at least 3 times greater stimulation of replicative DNA synthesis than non-nitrosated OiV. Non-nitrosated OiV itself induced scarcely any ornithine decarboxylase activity in the pyloric mucosa 16 h after its administration. The ornithine decarboxylase activity in the control group (pooled material from 3 rats) was 0.5 pmole CO2/30 min/mg protein. Doses of 1.0 and 2.0 g/kg body weight of OiV induced ornithine decarboxylase activities of 6.5 and 11.4 pmole CO2/30 min/mg protein. These increases were 1/10 of those induced by the nitrosated OiV fraction (Table 1). However, the mean value of 20 control groups up to the present is 5 _+4 pmole CO2/30 min/mg protein. The present results show that stimulation of replicative DNA synthesis and induction of ornithine decarboxylase after nitrosation of OiV are mainly caused by nitrosated OiV. Discussion
The present study demonstrated that oral administration of a nitrosated product of a crude ethanol extract from a medicinal plant, the OiV fraction, induced marked DNA single-strand scission, replicative DNA synthesis (RDS) and ornithine decarboxylase (ODC) activity in the pyloric mucosa of rat stomach. The administration of the nitrosated OiV fraction at doses of 2.0-2.8 g/kg body weight induced similar effects in rats to those of 10-100 mg/kg body weight of MNNG, a glandular stomach carcinogen (Furihata et al., 1987c, 1989a): (a) the elution rate constant (DNA singlestrand scission) was (23.5 × 10-3/ml)/(2.0 g nitrosated OiV fraction/kg body weight) vs. (28.8 × 10-3/ml)/(10 mg MNNG/kg body weight); (b) the RDS was (ll-fold increase)/(2.8 g nitrosated OiV fraction/kg body weight) vs. (17-fold increase)/(100 mg MNNG/kg body weight);
60
(c) the ODC activity was (546 pmole CO2/30 min/mg protein)/(2.0 g nitrosated OiV fraction/kg body weight) vs. (509 pmole CO2/30 min/mg protein)/(100 mg MNNG/kg body weight). Moreover, the nitrosated OiV fraction at doses of 1.4-2.0 g/kg body weight seemed to induce similar effects in rats to those of sodium chloride, a glandular stomach tumor promoter, at 1.0 g/kg body weight (Furihata et al., 1984a): (a) the RDS was (5-fold increase)/(1.4 g nitrosated OiV fraction/kg body weight) vs. (7-fold increase)/(1.0 g sodium chloride/kg body weight); (b) the ODC activity was (546 pmole CO2/30 min/mg protein)/(2.0 g nitrosated OiV fraction/kg body weight) vs. (656 pmole CO2/30 min/mg protein)/(1.0 g sodium chloride/kg body weight). These results suggest that the nitrosated OiV fraction has 1/200 of the possible tumor-initiating activity of MNNG, 1/20 of the possible tumor-promoting activity of MNNG and 1/2 of the possible tumor-promoting activity of sodium chloride. As the nitrosated OiV fraction is a crude fraction, further study is required to determine whether OiV contains a potent carcinogen precursor(s). 1-Nitrosoindole-3-acetonitrile (Wakabayashi et al., 1985), 3-diazo-N-nitrosobamethan (Kikugawa et al., 1987) and a nitrosated product of hickory smoke condensate (Ohshima et aI., 1989a) are direct-acting mutagens in Salmonella typhimurium TA98 and TA100 or in E. coli. They are formed after nitrosation of indole-3-acetonitrile (present in Chinese cabbage), bamethan (a cardiovascular drug) and hickory smoke condensate (a seasoning). They have been reported to induce RDS, ODC and DNA single-strand scission or UDS (Furihata et al., 1987a, 1988a; Ohshima et al., 1989b), suggesting that they may have tumorinitiating a n d / o r -promoting activities in the glandular stomach. The present material is the fourth nitrosated material found by the present method to show genotoxic and cell proliferative activity in the stomach mucosa in vivo. Intake of the bark of OiV as a traditional medicine is not very high in Thailand, being estimated to be less than 200 p.g/day (stated on the
label of packages produced by the Department of Medical Science, Ministry of Public Health of Thailand) and an ethanol extract of the bark contained 22% of the total dry weight. We used 500 mM nitrite as an optimal concentration to prepare nitrosated OiV. However, the nitrite concentration in the stomach is probably not more than about 1 raM. Thus although a nitrosation product(s) of a component(s) of OiV may be formed in the human stomach by interaction with nitrite, the amount of this toxic compound(s) formed is probably much less than 1 /xg/day, which is less than 10 6 of the doses used in the present study. Therefore, the relevance of nitrosated OiV to human cancers may be small. Ornithine decarboxylase is thought to be an enzyme related to cell proliferation. Previously we observed transient peaks of ornithine decarboxylase in the pyloric mucosa of rat stomach at various times during treatments with various glandular stomach carcinogens and tumor promoters. Sodium taurocholate induced a peak after 4 h (Furihata et al., 1987b), NaCI and catechol after 8-9 h (Furihata et al., 1984a, 1989b) and K2S205, formaldehyde, glyoxal and MNNG after 24 h (Furihata et al., 1985b, 1987c, 1988b, 1989c). In spite of these differences in the times of induction of maximal ornithine decarboxylase activity, all these chemicals induced maximal stimulation of replicative DNA synthesis 16-24 h after their administration.
Acknowledgements This work was done by Ms. Anong Tepsuwan during tenure of a fellowship from the JSPSNRCT Scientific Cooperation Program for Promotion of Science. We thank Mr. Kengo Hirose for technical assistance.
References Bose, P.K., and S.N. Bhattacharya (1938) Natural flavones. II. Coloring matters of the. bark of Oroxylum indicum Vent, J. Indian Chem. Soc., 15, 311-316. Burkill, I.H. (1935) A Dictionary of the Economic Products of the Malay Peninsula, Oxford University Press, Oxford, 1590 pp. Dhar, M.L., M.M. Dhar and B.N. Dhawan (1968) Screening
61 of Indian plants for biological activity, I, Indian J. Exp. Biol., 6, 232-247. Furihata C., Y. Sato, M. Hosaka, T. Matsushima, F. Furukawa and M. Takahashi (1984a) NaCI induced ornithine decarboxylase and DNA synthesis in rat stomach mueosa, Biochem. Biophys. Res. Commun., 121, 1027-1032. Furihata, C., Y. Yamawaki, S.S. Jin, H. Moriya, K. Kodama, T. Matsushima, T. Ishikawa, S. Takayama and M. Nakadate (1984b) Induction of unscheduled DNA synthesis in rat stomach mucosa by glandular stomach carcinogens, J. Natl. Cancer Inst., 72, 1327-1334. Furihata, C., Y. Sato, T. Matsushima and M. Tatematsu (1985a) Induction of ornithine decarboxylase and DNA synthesis in rat stomach by methylglyoxal, Carcinogenesis, 6, 91-94. Furihata, C., S. Yoshida and T. Matsushima (1985b) Potential initiating and promoting activities of diacetyl and glyoxal in rat stomach mucosa, Jpn. J. Cancer Res. (Gann), 76, 809-814. Furihata, C., Y. Sato, A. Yamakoshi, M. Takimoto and T. Matsushima (1987a) Inductions of ornithine decarboxylase and DNA synthesis in rat stomach mucosa by 1-nitrosoindole-3-acetonitrile, Jpn. J. Cancer Res. (Gann), 78, 432435. Furihata, C., R. Takezawa, T. Matsushima and M. Tatematsu (1987b) Potential tumor-promoting activity of bile acids in rat glandular stomach, Jpn. J. Cancer Res. (Gann), 78, 32-39. Furihata, C., S. Yoshida, Y. Sato and T. Matsushima (1987c) Inductions of ornithine decarboxylase and DNA synthesis in rat stomach mucosa by glandular stomach carcinogens, Jpn. J. Cancer Res. (Gann), 78, 1363-1369. Furihata, C., A. Yamakoshi, T. Matsushima, T. Kato and K. Kikugawa (1988a) Possible tumour-initiating and -promoting activities of 3-diazo-N-nitrosobamethan in rat stomach mucosa, Mutagenesis, 3, 299-301. Furihata, C., A. Yamakoshi and T. Matsushima (1988b) Induction of ornithine decarboxylase and DNA synthesis in rat stomach by formaldehyde, Jpn. J. Cancer Res. (Gann), 79, 917-920. Furihata, C., A. Hatta, Y. Sato and T. Matsushima (1989a) Alkaline elution of DNA from stomach pyloric mucosa of rats treated with glyoxal, Mutation Res., 213, 227-231. Furihata, C., A. Hatta, Y. Sato and T. Matsushima (1989b) Induction of ornithine decarboxylase and replicative DNA synthesis but not DNA single strand scission or unscheduled DNA synthesis in the pyloric mucosa of rat stomach by catechol, Jpn. J. Cancer Res. (Gann), 80, 1052-1057. Furihata, C., A. Yamakoshi, R. Takezawa and T. Matsushima (1989c) Various sodium salts, potassium salts, a calcium salt and an ammonium salt induced ornithine decarboxylase and stimulated DNA synthesis in rat stomach mucosa, Jpn. J. Cancer Res. (Gann), 80, 424-429. Galikov, P.P., and I.I. Brekhman (1967) The pharmacological investigation of the liquid extract from the bark of Oroxylum indicum, Rastit. Resur., 3, 446-451. Hell, A., and W. Zilling (1970) Reconstitution of bacterial DNA dependent RNA-polymerase from isolated subunits
as a tool for the elucidation of the role of the subunits in transcription, FEBS Lett., 11, 165-168. leamworapong, C., K. Kangsadalumpai and W. Rojanapo (1989) Mutagenicities of Thai folklore medicines and their nitrosation products, Fifth International Conference on Environmental Mutagens, 10-15 July, Cleveland, OH, p. 93. Kikugawa, K., T. Kato and Y. Takeda (1987) Formation of a highly mutagenic diazo compound from the bamethannitrite reaction, Mutation Res., 177, 35-43. Kissane, J.M., and E. Robins (1958) The fluorometric measurement of deoxyribonucleic acid in animal tissues with special reference to the central nervous system, J. Biol. Chem., 233, 184-188. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Mirvish, S.S. (1983) The etiology of gastric cancer: intragastric nitrosamide formation and other theories, J. Natl. Cancer Inst., 71,629-647. Ohshima, H., M. Friesen, C. Malaveille, I. Brouet, A. Hautefeuille and H. Bartsch (1989a) Formation of direct-acting genotoxic substances in nitrosated smoked fish and meat products: identification of simple phenolic precursors and phenyldiazonium ions as reaction products, Food Chem. Toxicol., 27, 193-203. Ohshima, H., C. Furihata, T. Matsushima and H. Bartsch (1989b) Evidence of potential tumour-initiating and tumour-promoting activities of hickory smoke condensate when given alone or with nitrite to rats, Food Chem. Toxicol., 27, 511-516. Perry, L.M., and J. Metzger (1980) Attributed properties and uses, in: Medicinal Plants of East and Southeast Asia, MIT Press, Cambridge, MA, 620 pp. Shah, R.C., C.R. Mehta and T.S. Wheeler (1936) The constitution of oroxylin-A, a yellow colouring matter from the root-bark of Oroxylum indicum Vent, part I, J. Chem. Soc. (London), 519-593. Sina, J.F., C.L. Bean, G.R. Dysart, V.I. Taylor and M.O. Bradley (1983) Evaluation of the alkaline elution/rat hepatocyte assay as a predictor of carcinogenic/mutagenic potential, Mutation Res., 113, 357-391. Subramanian, S.S., and A.G.R. Nair (1972a) Flavanoids of the stem bark of Oroxylum indicum, Curr. Sci., 41, 62-63. Subramanian, S.S., and A.G.R. Nair (1972b) Flavanoids of leaves of Oroxylurn indicum and Pajanelia longifolia, Phytochemistry, 11,439-440. Takeda, Y., and H. Kanaya (1982) A screening procedure for the formation of nitroso derivatives and mutagens by drug-nitrite interaction, Chem. Pharm. Bull., 30, 33993404. Wakabayashi, K., M. Nagao, T. Tahira, H. Sato, M. Katayama, S. Marumo and T. Sugimura (1985) 1-Nitrosoindole-3acetonitrile, a mutagen produced by nitrite treatment of indole-3-acetonitrile, Proc. Jpn. Acad. B, 61, 190-192.
Communicated by F.H. Sobels
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© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
MUTLET 00609
Genotoxicity and cell proliferative activity of a nitrosated Oroxylum indicum Vent fraction in the pyloric mucosa of rat stomach Anong Tepsuwan
1 Chie Furihata 2, Wannee Rojanapo 1 and Taijiro Matsushima 2
1 Biochemistry and Chemical Carcinogenesis Section, Research Division, National Cancer Institute, Bangkok (Thailand) and 2 Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Tokyo 108 (Japan)
(Received 15 February 1991) (Revision received 12 September 1991) (Accepted 13 September 1991) Keywords: Oroxylum indicum Vent; DNA single-strand scission; Ornithine decarboxylase;Replicative DNA synthesis; Rat stomach
mucosa
Summary 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 $9 mix to Salmonella typhimurium TA98 and TA100. Administration of the nitrosated OiV fraction at doses of 1 and 2 g / k g body weight induced dose-dependent D N A single-strand scission ( p < 0.02), determined by the alkaline elution method, in the stomach pyloric mucosa 2 h after its administration: a dose of 2 g / k g body weight induced an 18-fold increase in the D N A elution rate constant. Administration of the nitrosated OiV fraction at doses of 0.7-2.8 g / k g body weight also induced dose-dependent increases, up to ll-fold ( p < 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 / k g 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.
Oroxylum indicum Vent (OiV, commonly called Pay-gaa in Thailand) belongs to the family Bignoniaceae. It is a traditional medicinal plant in Thailand used in the treatment of various kinds of diseases, such as stomach disorders, diarrhea, and dysentery, as a diaphoretic (Burkill,
Correspondence: Dr. C. Furihata, Department of Molecular Oncology, Institute of Medical Science, Universityof Tokyo, Shirokanedai, Minato-ku, Tokyo 108 (Japan).
1935), and as a styptic and antipyretic for the treatment of rheumatic swellings (Perry and Metzger, 1980). It contains oroxylin A (5,7-dihydroxy-6-methoxyflavone), baicalein (5,6,7-trihydroxyflavone), chrysin and 6-methylbaialein (Shah et al., 1936; Bose and Bhattacharya, 1938; Subramanian and Nair, 1972a, b). The biological activities of this plant have been studied and results have shown that it has anti-inflammatory, antiallergic (Galikov and Brekhman, 1967) and antispasmodic (Dhar et al., 1968) activities. Crude ethanol and hexane extracts of this plant have no
56
mutagenic activity on Salmonella typhimurium TA98 or TA100 in either the presence or the absence of $9 mix. However, after nitrosation with nitrite under acidic conditions, these extracts become strongly mutagenic to Salmonella typhimurium strains TA98 and TA100 in the absence of $9 mix (Ieamworapong et al., 1989). These results suggest that a nitrosation product(s) in extracts of this plant is a direct-acting mutagen(s). A direct-acting mutagen/carcinogen produced by nitrosation in the acidic conditions of the stomach is thought to be a possible cause of human gastric cancer (Mirvish, 1983). Both tumor initiation and tumor promotion are thought to be important in human carcinogenesis. Therefore, the tumor-initiating and -promoting activities of the nitrosation product of OiV were examined to evaluate its potential hazard to humans. Furihata et al. (1984a, b, 1985a, 1989a) have developed in vivo short-term methods for evaluating possible tumor-initiating activity (genotoxicity) assessed as unscheduled DNA synthesis (UDS) and DNA single-strand scission determined by an alkaline elution method as markers, and also possible tumor-promoting activity (cell proliferative activity) using replicative DNA synthesis (RDS) and ornithine decarboxylase (ODC) as markers. In this study, we examined the possible tumor-initiating and -promoting activities of a nitrosated product(s) of OiV in the pyloric mucosa of rat stomach by these techniques after oral administration of an OiV fraction. The results indicate that the nitrosated product may have tumor-initiating and -promoting activities in the pyloric mucosa of rat stomach. Materials and methods
Preparation of nitrosated OiV fraction Dry bark of Oroxylum indicum Vent, purchased from a medicinal plant store in Bangkok, was chopped into small pieces and then blended to a powder. A crude ethanol extract was prepared by soaking the bark powder in 70% ethanol overnight and then filtering the mixture through Whatman No. 1 filter paper. The extraction process was repeated and the 2 filtrates were combined and evaporated to dryness at 55°C under
1000
800 c-
oo
600
o
% •-r
400
o
E
200
Z
0
I
0 20
I
I
I
50
100
200
Amount of OiV (IJg/plate) Fig. l. Appearance of mutagenicity to Salmonella typhimurium TA100 without $9 mix on treatment of OiV with nitrite. Nitrite concentrations 0 (X), 10 (o), 25 (zx), 100 (11), 250 ( o ) and 500 ( • ) raM.
reduced pressure in a rotary evaporator. The nitrosated product was prepared by a slight modification of the method of Takeda and Kanaya (1982). We tested nitrite concentrations of 10-500 mM (Fig. 1), and on the basis of the results we used 500 mM nitrite to prepare the nitrosated OiV fraction. Briefly, the ethanol extract was incubated at 37°C in the dark with 500 mM sodium nitrite in solution adjusted to pH 3 by addition of HC1. After incubation for 3 h, the mixture was centrifuged at 6000 × g for 15 min and the supernatant and the precipitate were separated. The supernatant was passed through a Sep-Pak C-18 cartridge to remove sodium chloride and sodium nitrite and also to concentrate the nitrosation product. The product was eluted from the cartridge with methanol and evaporated to dryness at 55°C in a rotary evaporator under reduced pressure. The precipitate that appeared after centrifugation was washed with cold distilled water and was dried in vacuo. The 2 fractions were combined and were used as the nitrosated OiV fraction. The mutagenicity of the samples was tested by the method of Maron and Ames (1983).
57
Animals and treatment Male Fischer rats ( F 3 4 4 / D u Crj: Charles River Japan, Inc., Kanagawa) 7-8 weeks old were given a limited amount of diet (4 g of commercial pellet diet (Nihon Clea, Tokyo) per rat of 200 g body weight) overnight to reduce their dietary stomach contents. The following day, they were given the nitrosated OiV fraction suspended in distilled, deionized water by gastric intubation. Control animals were given distilled, deionized water only. The stomach was removed at an appropriate time, opened along the greater curvature, and washed with sterile saline. The pyloric mucosa was scraped off with a razor blade or cut into small pieces with scissors and used for assays of DNA single-strand scission, RDS and ODC activity.
stomach was determined in in vitro organ culture in the presence of tritiated thymidine ([3H]dThd, 2.96 T B q / m m o l e , ICN Radiochemicals, Irvine, CA) as described previously (Furihata et al., 1984b, 1985a). The DNA fraction was extracted from the tissue and an aliquot was dissolved in ASC I1 (Amersham, Arlington Heights, IL). The incorporation of [3H]dThd into DNA was determined in a Packard Tricab 1500 liquid scintillation counter. The DNA contents of the DNA fractions were determined with 3,5-diaminobenzoic acid, with calf thymus DNA (Worthington Biochemicals Co., Freehold, N J) as a standard. Values for 5 individual rats were determind in each experiment. The results were analyzed statistically by Welch's test.
Alkaline elution of DNA
Ornithine decarboxylase (ODC) assay
A modification (Furihata et al., 1989a) of the method of Sina et al. (1983) was used for alkaline elution of DNA. The pyloric mucosa was obtained from rats 2 h after administration of the OiV fraction and lysed on a filter (Fluorinert, 1.0 /xm pore size and 25 mm diameter, Japan Millipore Ltd., Tokyo, Japan) with 3.0 ml of lysis solution (100 mM glycine, 25 mM E D T A and 2% sodium dodecylsulfate, pH 9.7) in the presence of proteinase K (1 m g / m l , Sigma Chemical Co., St. Louis, MO) for 60 min in the dark. After the lysis solution had passed through the filter, the filter was washed with 3 ml of a 2 mM E D T A solution, pH 10. Then DNA was eluted in the dark at 20-25°C with 30 ml of alkaline solution (20 mM E D T A and 1.95% tetra-n-propylammonium hydroxide, pH 12.1) at a flow rate of 0.05 ml/min. Fractions of 3.0 ml were collected and the DNA contents of the fractions of eluate and on the filters were determined with 3,5-diaminobenzoic acid (Tokyo Kasei Kogyo Co., Tokyo) (Kissane and Robins, 1958). The elution rate constant (ml-1) of D N A was calculated from a semi-logarithmic plot of the amount of DNA remaining on the filter (log scale) against the fraction number from the first to the fifth fraction. The results were analyzed statistically by Welch's test.
The mucosa of rat stomach was homogenized in ODC extraction buffer and ODC activity in the extract was determined using L-[1-~4C]ornithine (2.01 G B q / m m o l e , New England Nuclear, Boston, MA) as a substrate as described previously (Furihata et al., 1985a). Briefly, samples of pyloric mucosa from 3 rats were pooled and homogenized in 50 mM sodium phosphate buffer, pH 7.2, containing 0.1 mM pyridoxal phosphate and 0.1 mM ethylenediaminetetraacetic acid (EDTA). The homogenate was centrifuged at 16,000 rpm for 20 min at 4°C, and the supernatant was used for measurement of ODC activity. The protein contents of the extracts were determined by microassay as described by Heil and Zilling (1970) with bovine serum albumin as a standard. Results are shown as means for duplicate assays on pooled materials from 3 rats.
Repficative DNA synthesis (RDS) After administration of the nitrosated OiV fraction in vivo, RDS in the pyloric mucosa of the
Results
Induction of DNA single-strand scission Fig. 2 shows typical elution patterns of DNA obtained from the pyloric mucosa of control rats and those treated with the nitrosated OiV fraction. Fig. 3 shows the dose-dependent increase in the elution rate constant 2 h after administration of doses of 1 and 2 g / k g body weight. A dose of 2 g / k g body weight resulted in an 18-fold increase ( p < 0.02) in the elution rate constant. These results demonstrated that the nitrosated OiV
58
1.0
l
6000=
/..
3oooI ~{
g ==0.1
7< u_ ~ 0.05
Fracteon No
'
'0
I /
/':
"
o ,
Fig. 2. Typical alkaline elution patterns of D N A from the stomach pyloric mucosa of rats 2 h after treatment with the nitrosated OiV fraction at doses of 0 (HzO) (©), 1 ( A ) and 2 (•) g / k g body weight.
fraction caused significant induction of DNA single-strand scission in the pyloric mucosa of rat stomach after its oral administration at doses of 1 and 2 g / k g body weight.
0
0.7
1.4
2.8
Dose of nitrosated OiV fraction (g/kg body weight) Fig. 4. Dose-dependent increase in RDS in the pyloric mucosa 16 h after administration of the nitrosated OiV fraction. Values at each dose are for 5 individual rats. Points represent values for individual rats and bars show m e a n s of these individual values. Values were significantly different from control at doses of 1.4 g ( p < 0.02) and 2.8 g ( p < 0.05)/kg body weight by Welch's test.
g~ o
i 40
•
30 1
•
F
Stimulation of replicative DNA synthesis The nitrosated OiV fraction induced a dosedependent increase in RDS in the pyloric mucosa 16 h after its administration at doses of 0.7-2.8 g / k g body weight (Fig. 4). A dose of 2.8 g / k g body weight induced an ll-fold increase in RDS ( p < 0.05).
"
Induction of ornithine decarboxylase activity
Olqll
0
-
~
I
1
2
Dose of nitrosated OiV fraction (g/kg body weight) Fig. 3. Dose-dependent increase in elution rate constant of D N A from stomach pyloric mucosa of rats 2 h after administration of the nitrosated OiV fraction. Values at 1.0 and 2.0 g / k g body weight are significantly different from control by Welch's test ( p < 0.02).
Fig. 5 shows the induction of ODC activity in the pyloric mucosa of rat stomach with a maximum 4 h after administration of the nitrosated OiV fraction at a dose of 2 g / k g body weight. This maximum ODC activity, releasing 564 pmole C O z / 3 0 m i n / m g protein, was about 100-fold the control activity. A dose-dependent increase in ODC activity was induced in the pyloric mucosa 4 h after administration of the nitrosated OiV fraction at doses of 0.25-2.0 g / k g body weight (Table 1). We also observed a slight increase in ODC activity in the fundic mucosa with similar dose dependence as the activity in the pyloric mucosa. However, no
59 600 500 £ 400 E 300 cE 0 ~ 200 ,D
o 100 o c3 E O 0
I
I
I
4
8
16
I
24h
Time after administration Fig. 5. Induction of ODC activity in the pyloric mucosa of rat stomach by the nitrosated OiV fraction (2 g/kg body weight). Results are means for duplicate assays on pooled materials from the rats.
ODC induction was observed in the forestomach epithelium in a parallel experiment to that for the results in Table 1 (data not shown). These results suggest differences in the ability of the nitrosated OiV fraction to induce ODC activity in the pyloric mucosa, fundic mucosa and forestomach epithelium of rat stomach.
Stimulation of replicative DNA synthesis and induction of ornithine decarboxylase by non-nitrosated OiV Non-nitrosated OiV itself stimulated replicative DNA synthesis in the pyloric mucosa of rat
TABLE 1 DOSE-DEPENDENT INDUCTION OF ODC ACTIVITY IN THE PYLORIC MUCOSA OF RAT STOMACH BY THE NITROSATED OiV FRACTION Dose (g/kg body weight)
ODC activity (pmole CO 2/30 min/mg protein)
0 0.25 0.50 1.0 2.0
6.3 16.2 34.5 75.6 174.1
stomach to some extent. Replicative DNA synthesis in the untreated control group (4 rats) was 163 _+25 dpm [3H]dThd//xg DNA. Doses of 1.0 and 2.0 g / k g body weight of non-nitrosated OiV (4 rats in each group) induced replicative DNA synthesis of 307 _+42 (p < 0.001) and 365 _+ 124 (p < 0.05) dpm [3H]dThd//xg DNA, respectively. Thus the nitrosated OiV fraction caused at least 3 times greater stimulation of replicative DNA synthesis than non-nitrosated OiV. Non-nitrosated OiV itself induced scarcely any ornithine decarboxylase activity in the pyloric mucosa 16 h after its administration. The ornithine decarboxylase activity in the control group (pooled material from 3 rats) was 0.5 pmole CO2/30 min/mg protein. Doses of 1.0 and 2.0 g/kg body weight of OiV induced ornithine decarboxylase activities of 6.5 and 11.4 pmole CO2/30 min/mg protein. These increases were 1/10 of those induced by the nitrosated OiV fraction (Table 1). However, the mean value of 20 control groups up to the present is 5 _+4 pmole CO2/30 min/mg protein. The present results show that stimulation of replicative DNA synthesis and induction of ornithine decarboxylase after nitrosation of OiV are mainly caused by nitrosated OiV. Discussion
The present study demonstrated that oral administration of a nitrosated product of a crude ethanol extract from a medicinal plant, the OiV fraction, induced marked DNA single-strand scission, replicative DNA synthesis (RDS) and ornithine decarboxylase (ODC) activity in the pyloric mucosa of rat stomach. The administration of the nitrosated OiV fraction at doses of 2.0-2.8 g/kg body weight induced similar effects in rats to those of 10-100 mg/kg body weight of MNNG, a glandular stomach carcinogen (Furihata et al., 1987c, 1989a): (a) the elution rate constant (DNA singlestrand scission) was (23.5 × 10-3/ml)/(2.0 g nitrosated OiV fraction/kg body weight) vs. (28.8 × 10-3/ml)/(10 mg MNNG/kg body weight); (b) the RDS was (ll-fold increase)/(2.8 g nitrosated OiV fraction/kg body weight) vs. (17-fold increase)/(100 mg MNNG/kg body weight);
60
(c) the ODC activity was (546 pmole CO2/30 min/mg protein)/(2.0 g nitrosated OiV fraction/kg body weight) vs. (509 pmole CO2/30 min/mg protein)/(100 mg MNNG/kg body weight). Moreover, the nitrosated OiV fraction at doses of 1.4-2.0 g/kg body weight seemed to induce similar effects in rats to those of sodium chloride, a glandular stomach tumor promoter, at 1.0 g/kg body weight (Furihata et al., 1984a): (a) the RDS was (5-fold increase)/(1.4 g nitrosated OiV fraction/kg body weight) vs. (7-fold increase)/(1.0 g sodium chloride/kg body weight); (b) the ODC activity was (546 pmole CO2/30 min/mg protein)/(2.0 g nitrosated OiV fraction/kg body weight) vs. (656 pmole CO2/30 min/mg protein)/(1.0 g sodium chloride/kg body weight). These results suggest that the nitrosated OiV fraction has 1/200 of the possible tumor-initiating activity of MNNG, 1/20 of the possible tumor-promoting activity of MNNG and 1/2 of the possible tumor-promoting activity of sodium chloride. As the nitrosated OiV fraction is a crude fraction, further study is required to determine whether OiV contains a potent carcinogen precursor(s). 1-Nitrosoindole-3-acetonitrile (Wakabayashi et al., 1985), 3-diazo-N-nitrosobamethan (Kikugawa et al., 1987) and a nitrosated product of hickory smoke condensate (Ohshima et aI., 1989a) are direct-acting mutagens in Salmonella typhimurium TA98 and TA100 or in E. coli. They are formed after nitrosation of indole-3-acetonitrile (present in Chinese cabbage), bamethan (a cardiovascular drug) and hickory smoke condensate (a seasoning). They have been reported to induce RDS, ODC and DNA single-strand scission or UDS (Furihata et al., 1987a, 1988a; Ohshima et al., 1989b), suggesting that they may have tumorinitiating a n d / o r -promoting activities in the glandular stomach. The present material is the fourth nitrosated material found by the present method to show genotoxic and cell proliferative activity in the stomach mucosa in vivo. Intake of the bark of OiV as a traditional medicine is not very high in Thailand, being estimated to be less than 200 p.g/day (stated on the
label of packages produced by the Department of Medical Science, Ministry of Public Health of Thailand) and an ethanol extract of the bark contained 22% of the total dry weight. We used 500 mM nitrite as an optimal concentration to prepare nitrosated OiV. However, the nitrite concentration in the stomach is probably not more than about 1 raM. Thus although a nitrosation product(s) of a component(s) of OiV may be formed in the human stomach by interaction with nitrite, the amount of this toxic compound(s) formed is probably much less than 1 /xg/day, which is less than 10 6 of the doses used in the present study. Therefore, the relevance of nitrosated OiV to human cancers may be small. Ornithine decarboxylase is thought to be an enzyme related to cell proliferation. Previously we observed transient peaks of ornithine decarboxylase in the pyloric mucosa of rat stomach at various times during treatments with various glandular stomach carcinogens and tumor promoters. Sodium taurocholate induced a peak after 4 h (Furihata et al., 1987b), NaCI and catechol after 8-9 h (Furihata et al., 1984a, 1989b) and K2S205, formaldehyde, glyoxal and MNNG after 24 h (Furihata et al., 1985b, 1987c, 1988b, 1989c). In spite of these differences in the times of induction of maximal ornithine decarboxylase activity, all these chemicals induced maximal stimulation of replicative DNA synthesis 16-24 h after their administration.
Acknowledgements This work was done by Ms. Anong Tepsuwan during tenure of a fellowship from the JSPSNRCT Scientific Cooperation Program for Promotion of Science. We thank Mr. Kengo Hirose for technical assistance.
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61 of Indian plants for biological activity, I, Indian J. Exp. Biol., 6, 232-247. Furihata C., Y. Sato, M. Hosaka, T. Matsushima, F. Furukawa and M. Takahashi (1984a) NaCI induced ornithine decarboxylase and DNA synthesis in rat stomach mueosa, Biochem. Biophys. Res. Commun., 121, 1027-1032. Furihata, C., Y. Yamawaki, S.S. Jin, H. Moriya, K. Kodama, T. Matsushima, T. Ishikawa, S. Takayama and M. Nakadate (1984b) Induction of unscheduled DNA synthesis in rat stomach mucosa by glandular stomach carcinogens, J. Natl. Cancer Inst., 72, 1327-1334. Furihata, C., Y. Sato, T. Matsushima and M. Tatematsu (1985a) Induction of ornithine decarboxylase and DNA synthesis in rat stomach by methylglyoxal, Carcinogenesis, 6, 91-94. Furihata, C., S. Yoshida and T. Matsushima (1985b) Potential initiating and promoting activities of diacetyl and glyoxal in rat stomach mucosa, Jpn. J. Cancer Res. (Gann), 76, 809-814. Furihata, C., Y. Sato, A. Yamakoshi, M. Takimoto and T. Matsushima (1987a) Inductions of ornithine decarboxylase and DNA synthesis in rat stomach mucosa by 1-nitrosoindole-3-acetonitrile, Jpn. J. Cancer Res. (Gann), 78, 432435. Furihata, C., R. Takezawa, T. Matsushima and M. Tatematsu (1987b) Potential tumor-promoting activity of bile acids in rat glandular stomach, Jpn. J. Cancer Res. (Gann), 78, 32-39. Furihata, C., S. Yoshida, Y. Sato and T. Matsushima (1987c) Inductions of ornithine decarboxylase and DNA synthesis in rat stomach mucosa by glandular stomach carcinogens, Jpn. J. Cancer Res. (Gann), 78, 1363-1369. Furihata, C., A. Yamakoshi, T. Matsushima, T. Kato and K. Kikugawa (1988a) Possible tumour-initiating and -promoting activities of 3-diazo-N-nitrosobamethan in rat stomach mucosa, Mutagenesis, 3, 299-301. Furihata, C., A. Yamakoshi and T. Matsushima (1988b) Induction of ornithine decarboxylase and DNA synthesis in rat stomach by formaldehyde, Jpn. J. Cancer Res. (Gann), 79, 917-920. Furihata, C., A. Hatta, Y. Sato and T. Matsushima (1989a) Alkaline elution of DNA from stomach pyloric mucosa of rats treated with glyoxal, Mutation Res., 213, 227-231. Furihata, C., A. Hatta, Y. Sato and T. Matsushima (1989b) Induction of ornithine decarboxylase and replicative DNA synthesis but not DNA single strand scission or unscheduled DNA synthesis in the pyloric mucosa of rat stomach by catechol, Jpn. J. Cancer Res. (Gann), 80, 1052-1057. Furihata, C., A. Yamakoshi, R. Takezawa and T. Matsushima (1989c) Various sodium salts, potassium salts, a calcium salt and an ammonium salt induced ornithine decarboxylase and stimulated DNA synthesis in rat stomach mucosa, Jpn. J. Cancer Res. (Gann), 80, 424-429. Galikov, P.P., and I.I. Brekhman (1967) The pharmacological investigation of the liquid extract from the bark of Oroxylum indicum, Rastit. Resur., 3, 446-451. Hell, A., and W. Zilling (1970) Reconstitution of bacterial DNA dependent RNA-polymerase from isolated subunits
as a tool for the elucidation of the role of the subunits in transcription, FEBS Lett., 11, 165-168. leamworapong, C., K. Kangsadalumpai and W. Rojanapo (1989) Mutagenicities of Thai folklore medicines and their nitrosation products, Fifth International Conference on Environmental Mutagens, 10-15 July, Cleveland, OH, p. 93. Kikugawa, K., T. Kato and Y. Takeda (1987) Formation of a highly mutagenic diazo compound from the bamethannitrite reaction, Mutation Res., 177, 35-43. Kissane, J.M., and E. Robins (1958) The fluorometric measurement of deoxyribonucleic acid in animal tissues with special reference to the central nervous system, J. Biol. Chem., 233, 184-188. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Mirvish, S.S. (1983) The etiology of gastric cancer: intragastric nitrosamide formation and other theories, J. Natl. Cancer Inst., 71,629-647. Ohshima, H., M. Friesen, C. Malaveille, I. Brouet, A. Hautefeuille and H. Bartsch (1989a) Formation of direct-acting genotoxic substances in nitrosated smoked fish and meat products: identification of simple phenolic precursors and phenyldiazonium ions as reaction products, Food Chem. Toxicol., 27, 193-203. Ohshima, H., C. Furihata, T. Matsushima and H. Bartsch (1989b) Evidence of potential tumour-initiating and tumour-promoting activities of hickory smoke condensate when given alone or with nitrite to rats, Food Chem. Toxicol., 27, 511-516. Perry, L.M., and J. Metzger (1980) Attributed properties and uses, in: Medicinal Plants of East and Southeast Asia, MIT Press, Cambridge, MA, 620 pp. Shah, R.C., C.R. Mehta and T.S. Wheeler (1936) The constitution of oroxylin-A, a yellow colouring matter from the root-bark of Oroxylum indicum Vent, part I, J. Chem. Soc. (London), 519-593. Sina, J.F., C.L. Bean, G.R. Dysart, V.I. Taylor and M.O. Bradley (1983) Evaluation of the alkaline elution/rat hepatocyte assay as a predictor of carcinogenic/mutagenic potential, Mutation Res., 113, 357-391. Subramanian, S.S., and A.G.R. Nair (1972a) Flavanoids of the stem bark of Oroxylum indicum, Curr. Sci., 41, 62-63. Subramanian, S.S., and A.G.R. Nair (1972b) Flavanoids of leaves of Oroxylurn indicum and Pajanelia longifolia, Phytochemistry, 11,439-440. Takeda, Y., and H. Kanaya (1982) A screening procedure for the formation of nitroso derivatives and mutagens by drug-nitrite interaction, Chem. Pharm. Bull., 30, 33993404. Wakabayashi, K., M. Nagao, T. Tahira, H. Sato, M. Katayama, S. Marumo and T. Sugimura (1985) 1-Nitrosoindole-3acetonitrile, a mutagen produced by nitrite treatment of indole-3-acetonitrile, Proc. Jpn. Acad. B, 61, 190-192.
Communicated by F.H. Sobels