Fd Chem. Toxic. Vol. 29, No. 8, pp. 531-535, 1991 Printed in Great Britain

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HEPATIC O R N I T H I N E DECARBOXYLASE I N D U C T I O N BY POTATO GLYCOALKALOIDS IN RATS K. A. CALDWELL*,O. K. GROSJEANt, P. R. HENIKA and MENDELFRIEDMAN Food Safety Research Unit, Western Regional Research Center, Agricultural Research Service, USDA, Albany, CA 94710, USA (Accepted 18 April 1991) Abstract--The induction of hepatic ornithine decarboxylase (ODC) activity in rat livers by the potato glycoalkaloids ct-solanine,~t-chaconine,and their aglycone solanidine, has been studied. Ip administration of ~t-solanineat 7.5, 15 and 30 mg/kg body weight produced markedly elevated enzyme activity at 4 hr after treatment, with a linear dose response. The increase was four-fold at the lowest dose administered to 12-fold at the highest. ODC activity was measured at 1, 2, 3, 4, 5, 6, 8 and 24 hr after ~t-solaninewas given. A statistically significant increase in enzyme activity was evident at 3 hr after treatment; maximal activity occurred at 5 hr and was approximately 12 times greater than the dimethylsulphoxide (DMSO) control level. Elevated activities persisted for several hours, decreasing to about one-third of the maximal level at 8 hr. The relative effects of ~-solanine, ct-chaconineand solanidine on ODC activities were studied at 4hr using an equimolar dose of 17 mM/kg body weight. ODC activity induced by ~t-chaconinewas higher than that induced by ~-solanine; the latter activity was two-thirds that of the former. The aglycone solanidine did not induce any increase in activity compared with the DMSO control. ODC activity with dexamethasone, a glucocorticoid, at 4 mg/kg body weight, followed a pattern similar to that of ct-solanine. However, maximal activity occurred slightly earlier at 4 hr after treatment. The results show that the extent of induced ODC activity depends on the structure of the potato alkaloid.

INTRODUCTION Alkaloids occur naturally in many plants. They taste bitter and are toxic to man and mammals. The effects of acute or chronic exposure to alkaloids include neurotoxicity, hepatotoxicity, carcinogenicity and teratogenicity (Friedman, 1991; Morris and Lee, 1984). ct-Solanine and ct-chaconine (Fig. 1), two major glycoalkaloids in the potato, are synthesized in plants as a natural defence against insects and other pests. The toxicity of these alkaloids to humans is well documented (Harvey et al., 1985; McMillan and Thompson, 1979; Slanina, 1990) and has led to the implementation of a guideline limiting glycoalkaloid content to 20mg/100 g in a given potato cultivar. Based on a critical examination of the literature, Morris and Lee (1984) suggest that these guidelines may be too high. a-Solanine is poorly absorbed from the gastrointestinal tract of rats and is rapidly eliminated in the urine and faeces (Nishie et al., 1971). It is found in the spleen, kidney, liver, lung, fat, heart, brain and blood, ct-Chaconine is more toxic than a-solanine in mice following ip injection (Chaube and Swinyard,

*This paper is dedicated to the memory of Dr Kathryn A. Caldwell who died in the middle of this project. The work was carried out according to the plan she laid out before her untimely death. ?To whom all correspondence should be addressed. Abbreviations: DMSO = dimethylsulphoxide; ODC = ornithine decarboxylase; PMSF=phenylmethylsulphonyl fluoride; SGOT = serum glutamic-oxalacetic transaminase; SGPT = serum glutamic-pyruvic transaminase.

1976; Sharma et aL, 1979). Cardiotonic activities of various glycoalkaloids (Nishie et al., 1976) and the effect of ~t-solanine on blood-sugar level (Satoh, 1967) have been studied. Liver dysfunction was also examined (Dalvi, 1985) by administering ct-solanine orally and ip to male rats. However, no research has been conducted that uses ornithine decarboxylase (ODC) activity as a marker for hepatic cell proliferation (review by Kaczmarek and Kaminska, 1989) in response to glycoalkaloids. Since liver damage is a major endpoint of toxicity, there is a clear need for a reliable, rapid bioassay for quantifying hepatotoxicity or liver damage initiated by glycoalkaloids, especially in new potato varieties with high glycoalkaloid levels. Biochemical tests can serve this purpose and can also provide important clues about the chemical basis for the damaging effects. A biochemical test has major advantages over a test based on feeding animals a glycoalkaloid-containing diet in terms of duration, cost and required facilities. A possible disadvantage of a test based on ip injection is that the procedure bypasses digestion, absorption and transport processes, which may affect metabolism and utilization of the alkaloids. Russell and Snyder (I 969) showed that mammalian ODC possesses an extremely short half-life of only 10-20 rain in vivo. This property is certainly related to the mechanism of ODC inducibility. Also, the induction of ODC activity is highly correlated with the growth process, including all normal and pathological growth processes (Cohen, 1971; Rosenthal and Tabor, 1956; Russell, 1973). ODC is the initial enzyme in the biosynthesis of putrescine, spermine and spermidine (Janne et al., 1978). According to the

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Fig. 1. Structures of ~t-chaconine, ~t-solanine, solanidine and dexamethasone. study of O'Brien et al. (1975a,b) on tumour promotion in mouse skin, ODC induction was essential to the tumour promotion process, although other, undefined factors must also be involved. In an earlier paper, Caldwell (1987) was able to demonstrate dramatic increases in ODC induction in the rat pancreas, as stimulated by soy protein isolate. The objective of the present research was to help determine the hepatotoxicity of glycoalkaloids in rats by ip injecting ~t-solanine, ~t-chaconine and solanidine, and then measuring the in vivo activities of ODC in weanling rat livers. ODC activity was chosen to determine hepatotoxicity because ODC induction was suggested as an index of the amount of damage of a given agent on specific target tissues (Janne et al., 1978; Russell, 1985). MATERIALS AND METHODS Chemicals. L-[1J4C]ornithine (54.3 mCi/mmol or 52.0 mCi/mmol) was obtained from New England Nuclear (Boston, MA, USA). The ~-solanine, ~tchaconine, solanidine, L-ornithine, dexamethasone, dithiothreitol and HEPES were obtained from Sigma Chemical Co. (St Louis, MO, USA). Hyamine hydroxide (1 u in methanol) was obtained from US Biochemical Corp. (Cleveland, OH, USA). Phenylmethyisuiphonyl fluoride (PMSF) was obtained from Boehringer Mannheim Corp. (Indianapolis, IN, USA), pyridoxal phosphate from Calbiochem (San Diego, CA, USA) and coomassie protein assay reagent from Pierce (Rockford, IL, USA). • A n i m a l s . Male Sprague-Dawley rats (55-65 g, approx. 21 days old) were obtained from Simonsen Laboratories, Inc. (Gilroy, CA, USA). Rats were housed in wire cages and maintained in a controlled environment at 22°C with a 12-hr light/dark cycle. Rats received standard laboratory chow (Purina Labs, St Louis, MO, USA) and water a d lib. until 2 hr before treatment, ct-Solanine was administered ip in dimethylsulphoxide (DMSO), at doses ranging from

7.5 to 30.0 mg/kg body weight; 8 rats were used for each experiment. Two sets of controls of 2 rats each were used. One control group received DMSO at 5 mi/kg body weight and a second control group received dexamethasone at 4 mg/kg body weight. Four rats received doses as stated above of ct-solanine, ~t-chaconine or solanidine (4 rats/treatment group). The ~t-solanine, ct-chaconine and dexamethasone were dissolved in DMSO at room temperature, and solanidine was dissolved in DMSO in a boiling water-bath. O D C assay. Rats were killed by cervical dislocation. Livers were immediately removed, freed of visible fat, blotted and weighed. Minced suspensions of the tissue were homogenized in 4 volumes of ice-cold homogenizing buffer, consisting of 50 mMHEPES, pH 7.5, 5 mM-pyridoxal phosphate, 2mMdithiothreitol, 0.1mM-EDTA and 2.5mM-PMSF. Homogenization was accomplished in two 5-sec bursts using a small-probe Tekmar homogenizer at 0°C. Homogenates were then centrifuged at 100,000 g for 25 min at 4°C, and supernatants were stored at -80°C. These were assayed on the following day. Enzyme activity was defined as #mol CO2 released/mg protein/min. The ODC enzyme of liver homogenates was stabilized by inclusion of PMSF in the homogenizing buffer. Assays were performed in 18 x 60-mm test-tubes fitted with rubber stopper/centre well assemblies from Kontes Scientific Glassware (Vinland, N J, USA). Enzyme activity was determined by measuring the quantity of 14CO2 evolved and trapped in 100/~1 1 M-hyamine hydroxide. The substrate consisted of 0.075 #Ci L-[l14C]ornithine plus 1.67mM-L-ornithine.HCl contained in 150/~1 of homogenizing buffer. Enzyme reactions were initiated by adding 100/~1 of supernatant solution; reagent blanks received 100#1 of homogenizing buffer. Assays were carried out with constant shaking for 60 min at 37°C. To terminate enzyme action, 300 #1 40% trichloroacetic acid was added by injection through the rubber stopper, and

ODC induction by potato glycoalkaloids incubation was continued for an additional 20 min. Centre wells and their contents were transferred to scintillation vials for radioactivity counting in 10 ml of a toluene-based scintillant. Radioactive standards, consisting of 15#1 of substrate solution and 100/~1 hyamine hydroxide, were also counted as above. Reagent blanks and radioactive standards were determined in triplicate; enzyme assays were performed in duplicate. The protein content of the supernatants was measured according to Bradford (1976); bovine plasma y-globulin served as the protein standard. Statistics. Statistical evaluation was carried out using a two-tailed t-test. Values were considered significant if P < 0.01 (see Results for explanation). RESULTS The effect of ~-solanine on the activity of O D C in weanling rat livers was examined first. Table 1 shows time-dependent changes in O D C activity in response to ct-solanine and dexamethasone. O D C activity increased significantly as early as 3 hr after administration of ~t-solanine at a dose of 30 mg/kg body weight and dexamethasone at a dose of 4 mg/kg body weight. Since no significant time-dependent change was detected in D M S O controls, all these values were averaged. The mean D M S O control values were 49.6 ___7.6nmol CO2/mg protein/min. The increase was greatest at 5 hr after treatment with ct-solanine, and the relative increase varied from five-fold at 3 hr to 12-fold at 5 hr. Elevated activities persisted for several hours, decreasing to about one-third of the maximal level at 8 hr, and declined to near control level by 24 hr. The maximal activity occurred at 4 hr after treatment with dexamethasone, with a 12-fold increase. This decreased sharply to less than half of the maximal activity at 5 hr and remained at that level until 24 hr. The statistical evaluation was done using a twotailed t-test. In almost all cases the changes were found to be statistically significant at the 99% confidence level (P < 0.01). The only times P values were greater than 0.01 were in the 0 - l - h r interval for both ~t-solanine and dexamethasone, and in the 4-5-hr interval for ct-solanine. However, even in these cases the P values were less than 0.05. While P < 0.05 is still a good statistical probability, it leaves some questions about any possible induction period in the

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first hour and about the actual peak position with -solanine between 4 and 5 hr. These fine details do not affect our conclusions. The dose dependence of O D C activity resulting from ~t-solanine is shown in Fig. 2. Within the range of ~t-solanine tested from 7.5 to 30.0mg/kg body weight at 4 h r , the response was approximately linear throughout. The increase ranged from fourfold at the lowest dose administered to 1 l-fold at the highest. Table 2 compares the effects of ~-solanine, ctchaconine and solanidine on O D C activity. An equimolar dose of 17mM/kg body weight, which is comparable to 15 mg ct-solanine/kg body weight, was administered ip and rats were killed 4 hr after treatment. ~t-Chaconine showed higher O D C activity than ct-solanine, and solanidine did not show any considerable increase in activity compared with the D M S O control.

Table I. Relative effects of time on hepatic ODC activity in ~t-solanine-and dexamethasone-treated rats ODC +SEMt (sp. act. × 104) Fold increase Hr after d o s i n g :t-Solanine:~ Dexamethasone~ :t-Solanine Dexamethasone Control (DMSO) 49.6 _+7.6 49.6 + 7.6 I 45.1 _+6.1" 36.4_+2.9* 0.9 0.7 2 89.3 ___10.7 78.9 + 20.9 1.8 1.6 3 248.2 ± 9.0 247.3 _4-58.8 5.0 5.0 4 536.5 __+21.4 594.3 -+ 55.4 10.8 12.0 5 591.8 + 59.4* 241.7 _+16.2 11.9 4.9 6 361.7 _+53.8 217.2 -+ 13.5 7.3 4.4 8 206.8 _+23.3 176.1 + 49.3 4.2 3.6 24 82.6 + 2.3 150.5 + 52.1 1.7 3.0 tSp. act. is defined as ~mol CO2 released/rag protein/rain. ~/~-Solanine was administered at 30 mg/kg body weight. §Dexamethasone was administered at 4 mg/kg body weight. Values are means _+SEM for 8 rats/group (ot-solanine)or 2 rats/group (dexamethasone) and those marked with asterisks are significantly different (two-tailed t-test) from the corresponding DMSO control value (*P < 0.05; for all other values P < 0.01). F(yr 29/~B

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K. A. CALDVdELLet al.

534

Table 2. Relativeeffectsof variousglycoalkaloidson hepatic ODC activityin rats* Glycoalkaloid(17 mr,l) ODC __.SEMt (sp. act. × 104) Control (DMSO) 49.6 +_.7.6 ~t-Solanine 359.2 __.81.0 ~t-Chaconine 561.8 +__31.4 Solanidine 93.0 + 27.6 *An equimolardose of 17mM/kgbody weightwas administered ip and rats were killed4 hr after treatment. Values are means+ SEM for duplicate determinations of 4 rats/group. fSp. act. is definedas/tmol CO2 released/mgprotein/min. DISCUSSION This study shows that ~-solanine, ct-chaconine and dexamethasone, administered ip, are major stimuli of liver ODC activity. They produced an early and pronounced increase after treatment, and the levels induced were typical of those produced in other organs by other stimuli, such as anterior pituitary polypeptide tropic hormones and steroid hormones (Russell, 1980). In most systems, ODC activity in response to stimuli is greatest about 4 hr post-treatment, returning to basal levels about 2 hr later (Janne et al., 1978; Russell, 1980; Tabor and Tabor, 1972). In this study, the enzyme activity peaked at 5 hr and remained elevated for at least 8 hr after ~t-solanine administration. Since our results with dexamethasone, which was used as a positive control, were comparable with those of ~-solanine, we will review briefly the reported effects of various hormones on ODC activity to place our findings in proper perspective (Janne et al., 1978; Russell, 1985). Panko and Kennedy (1971) examined the effect of a variety of hormones on hepatic ODC activity in adrenalectomized rats. Nearly all those treated showed increased activity to some extent, but the most effective hormone was hydrocortisone, the adrenal steroid, which induced a maximal activity at 3 hr after the first hormone injection, cAMP is known to be a mediator of many hormonal responses and has been shown to increase the activity of a number of hepatic enzymes in vivo. Beck et al. (1972) showed that the hepatic ODC activity of rats was stimulated more than seven-fold 3 hr after a single ip injection of dibutyryl cAMP. Theophylline or dexamethasone drugs, which may increase endogenous levels of cAMP, also increased ODC activity 10- and 21-fold, respectively. These results suggest that cAMP may be involved in the stimulation of hepatic ODC activity, possibly through the formation of new messenger RNA. Rapid and substantial elevation in ODC levels has been linked to tumour promotion, as in the case of the induction of ODC by 12-O-tetradecanoylphorbol-13-acetate (O'Brien et al., 1975a,b; Russell, 1980 and 1985; Russell and Snyder, 1969). In our present study, we administered an ip injection of 4 mg dexamethasone/kg body weight, which was the amount used in the study of Bishop et al. (1985). The dexamethasone-induced enzyme activity in rat liver followed the same pattern as with ct-solanine. Since liver damage is usually accompanied by a decrease in serum cholinesterase (Vorhaus et al., 1951), hepatic dysfunction in male rats was studied by Dalvi (1985) by measuring cholinesterase, serum glu-

tamic-oxalacetic transaminase (SGOT) and serum glutamic-pyruvic transaminase (SGPT) activities after administering solanine orally and ip. According to Dalvi's results, solanine (20 mg/kg body weight) administered ip is more toxic to liver than ingested solanine (250 mg/kg), which suggests a slow absorption of solanine in the gastro-intestinal tract. The increase in SGOT and SGPT activities and the inhibition of cholinesterase activity seen in the Dalvi study, following an ip injection of solanine, confirm that solanine is a hepatotoxic compound. However, the inhibition of cholinesterase by ~t-solanine was less than twice that of the control. The magnitude of increase in ODC activity observed in the present research by administration of ct-solanine is comparable with the induction caused by dexamethasone. At 7.5 mg/kg body weight of weanling rats, approximately one-ninth of the LDs0, and about one-third of the amount Dalvi (1985) used, ODC activity increased four-fold at 4 hr using ip injection. The present study of the induction of ODC activity by different potato glycoaikaloids shows that ~tchaconine is more potent than ct-solanine, indicating that the nature of sugars and not of the aglycone may be responsible for enzyme induction. This is contrary to the conclusion of Nishie et al. (1976) in their study using frog heart. As shown by our results with solanidine, the presence of sugars is critical for inducing ODC activity. In conclusion, our data demonstrate for the first time that Solanum glycoalkaloids induce ODC activity in rat livers. The extent of induction is similar to that previously reported for steroidal hormones such as dexamethasone, and is influenced by the nature of the carbohydrate attached to the 3-OH position of the aglycone part of the glycoalkaloid. ODC induction and measurement may provide a bioassay for the in civo biological activity of these glycoalkaloids that is quantitative and relatively short term. Only mg quantities of test material are required, and the results are available in 2 days. The magnitude of liver ODC induction by potato glycoalkaloids and the sustained nature of the increase suggest the possibility that these changes may not only relate to an adaptive response to ip injection. Further research is required to clarify the nature and mechanism of potato glycoalkaloid action. Acknowledgements--We thank Dr A. E. Pavlath for encour-

aging the completion of this work, and for development of the program used for statistical analyses. We also thank Dr J. C. Zahnley for helpful discussions. REFERENCES

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Hepatic ornithine decarboxylase induction by potato glycoalkaloids in rats.

The induction of hepatic ornithine decarboxylase (ODC) activity in rat livers by the potato glycoalkaloids alpha-solanine, alpha-chaconine, and their ...
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