Introduction

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The extracts of the flowers and leaves of Silybum marianum (St. Mary's thistle) have been used for centuries to treat liver diseases. In 1960, Janiak and Hänsel (1960) isolated the biological active principles of the extracts, and the chemical structures were elucidated by Pelter and Hänsel (1968), as well as by Wagner et al. (1968, 1971). The isolation led first to a mixture that was named silymarin, and it was with this mixture that most of the clinical studies were carried out. The main constituents are silibinin, isosilibinin, silicristin, and silidianin (Fig. 1).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Most important components of the silymarin mixture (silibinin formerly called silybin).

Most of the clinical findings can be summarized by the term "increased liver regeneration". Fintelmann and Albert (1980) observed that after toxic liver damage, the administration of silymarin resulted in a significantly accelerated normalization of serum glutamate-oxalacetate-transaminase and glutamate-pyruvate-transaminase values.

A double-blind study by Feher (1989) demonstrated that after administration of silymarin to patients with alcohol-induced liver disease, a distinctly more rapid normalization of the transaminases and bilirubin takes place. After treatment with silymarin, Berenquer and Carrasco (1977) observed a faster normalization of the plasma albumin values in patients with chronic inflammatory liver diseases. A multicenter study by Ferenci and colleagues (1989) in Vienna showed that the average survival rate of cirrhotic patients in the Child A stage could be markedly lengthened by silymarin.

Numerous investigations into the biochemical mechanism of action of silymarin have been performed by Sonnenbichler et al. It was confirmed that the transcription process in the livers of rats and mice in vivo (Sonnenbichler et al., 1976) is accelerated under the influence of silymarin and purified silibinin. In in vitro experiments with isolated cell nuclei and nucleoli, Machicao and Sonnenbichler (1977) demonstrated that the enzymatic activity of DNA-dependent RNA-polymerase I is stimulated by silymarin. Sonnenbichler and Zetl (1984) subsequently found that silibinin accelerates the synthesis of 28S, 18S, and 5.8S ribosomal RNAs and also promotes the formation of complete ribosomes. Thus, protein biosynthesis is indirectly intensified (Sonnenbichler and Zetl, 1986). Stimulation of RNA and protein biosynthesis was actually found to result in an increase in the rate of DNA biosynthesis in partially hepatectomized rat livers (Sonnenbichler et al., 1986). In these experiments, no significant difference was observed, irrespective of whether silibinin or the silymarin mixture was used. These findings provide biochemical confirmation of the clinical data on accelerated cell regeneration.

In view of this biochemical mechanism, silibinin was expected to exhibit a similar effect on other cell types. The effect on kidney cells was of particular interest, as these are frequently damaged by analgesics, cytostatics, and other drugs. For this reason, the extent to which silibinin influences the molecular biology of kidney cells was examined. The question as to whether silibinin is capable of antagonizing toxin-induced damage was of particular interest. To this end, nephrotoxins such as paracetamol, cisplatin, and vincristine were used to examine the effect of silibinin applied before or after the chemical-induced injury. It was discovered in earlier experiments (Andres, 1996), that of the four most important flavonolignans of the silymarin mixture (Fig. 1), only silibinin and silicristin have a stimulating effect on transcription and translation in liver cells. This was also to be tested in the case of kidney cells. Two nonmalignant cell lines from the kidneys of African green monkeys were selected for these experiments to warrant optimal reproducibility, to minimize statistical problems, and to follow ethical aspects concerning animal experiments. Proliferation rate, biosynthesis of DNA and protein, and the activity of the enzyme lactate dehydrogenase (LDH; as a measure of the cellular metabolic activity) were taken as the parameters defining the effect of the silymarin components.

	Materials and Methods

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Cell Cultures. All experiments were performed under sterile conditions. The adherent permanent cell lines from the kidneys of Cercopithecus aethiops (African green monkey): Vero: ATCC CCL 81 (passages 160 to 180) and BSC-1: ATCC CCL 26 (passages 176 to 182) were cultivated in 15 ml of Medium 199 (Gibco BRL, Life Technologies, Inc., Berlin, FRG) with Earle's salt, 2.2 g/l NaHCO₃, L-glutamine, T-tryptophan, and 5% fetal bovine serum (Gibco) in culture flasks (Falcon; Becton-Dickinson, Heidelberg, FRG) lying horizontally in an incubator (type B5060EK-CO₂; Heraeus, Hanau, FRG) in a moist atmosphere at 37°C with a gas mixture of 5% CO₂/95% air (5.1 × 10⁴ cells/ml, growth area 75 cm²). Seven days after the transfer, the media were renewed and the cells were transferred after an additional 7 days. To this end, after removal of the medium, the cells were treated according to Lindl and Bauer (1989) with 1.5 ml of 0.05% trypsin plus 0.02% EDTA for approximately 30 min at 37°C. Then 4 ml of Medium 199 and 5% fetal bovine serum were added.

Determination of Vitality. In all cases, the cells were counted after trypsination and the vitality determined according to Widholm (1972) by means of staining with fluorescein diacetate (0.5% w/v) in acetone.

Determination of Proliferation Rates. In each case, the cells were transferred in 400 µl to the wells at a concentration of 7.5 × 10⁴ cells/ml. Trypsination followed after 0, 1, 3, 4, 5, 6, and 7 days and counting was performed in a Fuchs-Rosenthal chamber (Brand GmbH, Wertheim, FRG) to determine the proliferation rates. The cells were prepared, and 24 h later the test substances were added in different concentrations to determine their effects on the cells.

Measurement of DNA Biosynthesis. The cells were transferred to wells at a concentration of 7.5 × 10⁴ cells/ml. After 24 h, the flavonolignans were added at concentrations of 20 and 100 µM, followed immediately by 0.5 µCi/ml of [methyl-¹⁴C]thymidine (specific activity 51 mCi/mmol). Trypsination was performed after 0, 24, 48, and 72 h, whereupon 2 × 100-µl samples were taken, to which 250 µl of ice-cold 0.066 M thymidine solution were pipetted. After 10 min, 5 ml of cold 10% trichloroacetic acid (TCA) and 0.02 M thymidine were added. After the samples had been allowed to stand on ice for at least 30 min, they were filtered through nitrocellulose filters (pore size, 0.2 µl; diameter, 25 mm; Sartorius, Göttingen, FRG) that had been preincubated in 10% TCA with 0.02 M thymidine. Then each sample was washed with 20 ml of 10% and 30 ml of 5% TCA solution (both with 0.02 M thymidine), and the filters were transferred to scintillation vessels and dried for 30 min at 60°C. After dissolution of each filter in 10 ml of scintillation liquid Aquasol-2 (DuPont-NEN, Dreieich, FRG), the samples were shaken thoroughly (gyratory shaker; New Brunswick Scientific Co., Inc., Edison, NJ) for 30 min, and the radioactivity was determined.

Measurement of Protein Biosynthesis. Immediately after dissemination (7.5 × 10⁴ cells/ml in well), the flavonolignans were added to the cells at the appropriate concentrations (0, 10, 20, 60 µM). The cells were incubated for 72 h, and then 0.125 µCi/ml of sample [2-¹⁴C]leucine (specific activity, 310 mCi/mmol) were added. Trypsination was performed after 0, 4, 8, 12, and 28 h, whereupon 2 × 100-µl samples each were taken to which 50 µl of cold 0.012 M L-leucine solution were added. After an additional 10 min, the proteins were precipitated with 5 ml of ice-cold 10% TCA (with 0.02 M L-leucine). Cleavage of the amino acid-carrying RNA was induced by heating the samples to 85°C for 35 min in a drying cabinet, cooling, and then filtering through nitrocellulose filters that had been incubated in 10% TCA with 0.02 M L-leucine solution before filtration. Additional processing is as described above.

Determination of LDH Activity (Stevens et al., 1986). The test substances were added to the cells 24 h after transfer (7.5 × 10⁴ cells/ml in well). With gentle shaking, the cells were suspended for 5 min with 0.1% Triton X-100 after an additional 0, 24, 48, and 72 h; then 400 µl of Medium 199 was added, and the cells were centrifuged for 6 min (Eppendorf centrifuge) before being stored on ice. After the addition of 1 ml of 0.1 mM nicotine adeninedinucleotide (NADH) + 1 mM pyruvate in 0.2 M tris(hydroxymethyl)-aminomethane-HCl (pH 7.3), the LDH activity in 10 µl samples was measured by determining the decrease in extinction at 340 nm for 20 min and calculating E/min. The molar extinction coefficient  of NADH at a layer thickness d of 1 cm at 340 nm is 6.22 cm²/µmol.

Tests. For the purpose of determining the growth curves and the protein and DNA synthesis and for measuring the LDH activities, 7.5 × 10⁴ cells/ml were cultivated in wells (growth area 2 cm²) of cell cluster (Costar, Bodenheim, FRG) each in 400 µl medium. Removal of the medium was followed in each case by washing with 100 µl 0.15 M NaCl, adding 100 µl of trypsin cocktail, heating to 37°C for 20 min, and then adding 100 µl of Medium 199 + 5% fetal bovine serum.

Statistics. Each value of the tables and figures was determined at least 8-fold. To characterize the experimental significance, the S.D.s and the statistical probabilities were calculated using Student's t distribution (Meyer, 1975).

Application of Test Substances. To improve solubility, the water-soluble silibinin hemisuccinate was used in the majority of experiments. In the experiments comparing silibinin with silicristin, silidianin, and isosilibinin, unesterified silibinin and the other flavonolignans were added after dissolution in ethanol owing to the greater solubility. Consequently, the final concentration of ethanol in the cell cultures was 1%. The controls were performed without ethanol and with 1% ethanol.

Damage by Toxicants and Administration of Silibinin. For subsequent administration after the chemical induced injury, 7.5 × 10⁴ cells/ml were incubated in the wells. The toxicants were added to the cells 23 h after dissemination as shown in Fig. 2. When the cells were exposed to the toxicants for a period of 60 min, they were washed 3 times each with 100 µl of Medium 199. This was followed by the addition of 400 µl of Medium 199 with 5% bovine serum, followed immediately by silibinin hemisuccinate in various concentrations (generally 20 and 40 µM) and radioactive precursor, where necessary. Then the treatment was continued as described above.

View larger version (8K): [in this window] [in a new window]   Fig. 2.   Treatment of the cells with administration of the flavonolignans after the chemical-induced injury.

View larger version (7K): [in this window] [in a new window]   Fig. 3.   Treatment of the cells with administration of the flavonolignans before the chemical-induced injury.

	Results

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In the present study, two adherent permanent cell lines from the kidneys of the African green monkey (C. aethiops) were selected: fibroblast-like Vero and epitheloid BSC-1 cells.

The growth curves in culture flasks show the typical sigmoid course with a resting phase of approx. 2 days followed by an exponential increase in cell count for up to 8 days in the case of Vero cells and with a resting phase of approx. 1 day and an exponential increase in cell count for up to 7 days for BSC-1 cells. The cells double approx. every 24 h before the stationary phase with a cell count of 6.3 × 10⁶/ml. The cultures exhibit confluent growth in contrast to malignant cells. Both cell lines show similar behavior in the wells of the culture clusters. The logarithmic phase has a duration of approx. 5 days, after which the growth curves reach a plateau with a cell count of 1.8 × 10⁶/well. Culture clusters were used exclusively for testing the substances because they allow for eight samples per experiment in multiple parallel batches.

Effect of Silibinin on the Proliferation of Vero Cells. Silibinin hemisuccinate (10 µM) was added to the cells and the proliferation rate monitored for a period of 5 days, with constant measurement of the vitality. In comparison to the controls, an increase in growth of 14% is observed 3 days after addition of silibinin (Table 1). After 5 days, this stimulation remains as high as 12% at constant vitality (95 to 96%), and the cells have reached the stationary phase. It is evident from this experiment that silibinin has a distinct stimulatory effect on the growth of Vero kidney cells.

Effect of Silibinin on DNA Biosynthesis in Vero Cells. The DNA biosynthesis was measured over a period of 72 h. Twenty-four hours after dissemination, varying quantities of silibinin hemisuccinate were added to the Vero cells followed shortly by [¹⁴C]thymidine. Seventy-two hours after the addition of 20 µM silibinin hemisuccinate, the DNA biosynthesis is found to increase by 19%, and the addition of 100 µM silibinin hemisuccinate again results in an 8% inhibition of precursor incorporation in newly synthesized DNA, comparable to the inhibition observed in the proliferation measurements.

Effect of Silibinin on Protein Biosynthesis in Vero Cells. The protein biosynthesis was determined at intervals by measuring the nonhydrolyzable radioactivity in the TCA precipitates of the high-molecular cell constituents after incorporation of radioactive leucine. The protein biosynthesis in cells treated with 20 µM silibinin hemisuccinate is stimulated by 16% after 24 h and by 21% with 40 µM (Table 1). Increasing the concentration to 60 µM silibinin hemisuccinate does not affect protein biosynthesis. Evidently, this is the threshold concentration for the above-mentioned inhibition. At 100 µM, the incorporation drops to 90% of that of the controls.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Time-dependent incorporation of [2-14C]leucine into newly synthesized proteins of Vero kidney cells in the presence of 20 µM flavonolignans. The statistical significance of the values (n = 8) at 48 h are calculated in comparison with the corresponding control (). Silibinin (), silicristin (), silidianin (), and isosilibinin (*); p for all <.001.

Effect of Silibinin on BSC-1 Cells. BSC-1 cells failed to exhibit a stimulation by 10 µM silibinin hemisuccinate, yet a growth increase of up to 11% was observed 4 days after the addition of 20 µM silibinin hemisuccinate and 19% with 40 µM (Table 1). With regard to protein biosynthesis, too, stimulation is at first less pronounced and only approximates the Vero cell values at concentrations of 40 µM silibinin hemisuccinate (Table 1). Again, the inhibition threshold occurs at concentrations >60 µM. For this reason, we decided to focus our further work on the more active Vero line.

Effects of Additional Flavonolignans from the Milk Thistle on Vero Kidney Cells. In our department, the four flavonolignans were tested in transcription and translation systems with liver cells (Andres, 1996). These investigations indicated that it could be pertinent to test these substances on kidney cells. For reasons of solubility, the flavonolignans silidianin, silicristin, isosilibinin (not available as hemisuccinate), and pure silibinin were dissolved in ethanol for these experiments and added to Vero cells at a concentration of 20 µM after dissemination. The final ethanol concentration in the test mixture was 1%, a concentration at which ethanol has no detectable effect on the parameters measured in the cells.

Effects on Proliferation. Flavonolignans (20 µM) were added to Vero cells 24 h after transfer. The cells were counted 72 h later and their vitality determined. The measured values are listed in Table 2. The data obtained show a 16% increase in growth rate after addition of silibinin. A 14% stimulation of proliferation was also observed in the case of those cells treated with silicristin. On the other hand, the flavonolignans silidianin and isosilibinin show slight to moderate inhibitory effects compared with the controls (6 and 10%, respectively).

Protein Biosynthesis under the Influence of Silibinin, Silicristin, Silidianin, and Isosilibinin. Twenty-four hours after addition of the flavonolignans, [2-C¹⁴]leucine was added to the culture clusters. Figure 4 shows the leucine incorporation into proteins of Vero cells under the influence of flavonolignans as a function of time.

Effects of Flavonolignans on the LDH Activity of Vero Kidney Cells. To determine the effect of flavonolignans on the LDH activity of cells, silibinin, silidianin, silicristin, and isosilibinin were added to the cells at a concentration of 20 µM 24 h after transfer. The LDH activity was determined over a period of 72 h. Figure 5 shows the change in activity with and without flavonolignans as a function of time.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Time-dependent LDH activities in Vero kidney cells under the influence of 20 µM flavonolignans. The statistical significances (in parentheses) of the values (n = 8) at 48 (A) and 72 (B) h are calculated in comparison with the corresponding control (). Silibinin (; A, p < .001, B, p < .001), silicristin (; A, p < .001, B, p < .001), silidianin (; A, p < .05, B, p < .001), and isosilibinin (*; A, p < .001, B, p < .001).

Effect of Silibinin on Vero Kidney Cells Damaged by Various Toxicants. In preceding experiments, the concentration dependence of the toxicity of paracetamol, cisplatin, and vincristine was examined to achieve suitable inhibitions of cell activities in the following examinations. The cell activities in the absence of toxicants were taken as control values. Because those cells treated with toxicants in the experiments on the postadministration of silibinin (after the chemical-induced injury) were exposed to the toxicants for only 1 h (Fig. 2), whereas they were exposed constantly in the case of previous administration of silibinin (Fig. 3), paracetamol and vincristine must be present in different concentrations in the experiments. The permanent presence of toxicants in the wells of the culture clusters implies that the toxicant concentration may not be as high as when the duration of exposure is only 1 h. This does not apply to experiments with cisplatin, in which the same cisplatin concentrations could be used irrespective of whether administration of silibinin was before or after the chemical injury, because in addition, cisplatin exerts its effect on kidney cells instantly and fully (blocking of DNA biosynthesis).

Paracetamol and Administration of Silibinin after the Chemical-Induced Injury. To examine the effect of silibinin after damage by paracetamol, the proliferation rates, protein biosynthesis, and LDH activity were measured again. It can be seen from the data in Table 3 that 6.62 mM paracetamol inhibits the proliferation rate by 18%. The addition of 20 µM silibinin hemisuccinate reduces this inhibition to 3% and results in growth similar to that of untreated controls. Silibinin hemisuccinate on its own increases the growth rate as described above.

Paracetamol and Administration of Silibinin before the Chemical-Induced Injury. The following experiments examined the extent to which the previous administration of silibinin is capable of lowering the inhibition of proliferation resulting from paracetamol in Vero kidney cells. In this series of experiments, lower paracetamol concentrations were selected because paracetamol was permanently present in the culture clusters.

Cisplatin and Administration of Silibinin after the Chemical-Induced Injury. After cisplatin damage induced with 8.4 µM, measurements showed a decrease in growth of approx. 34%, which was reduced to 19% on addition of 20 µM silibinin hemisuccinate and to 13% on addition of 40 µM (not listed in Table 5).

Cisplatin and Administration of Silibinin before the Chemical-Induced Injury. Despite the inevitable differences in experimental procedure with the permanent presence of toxicants, it was possible to work with cisplatin concentrations similar to those of the experiments with subsequent application of silibinin.

Vincristine and Administration of Silibinin after the Chemical-Induced Injury. To determine the effect with the flavonolignan silibinin after damage by vincristine, the proliferation, protein biosynthesis, and LDH activity were measured again.

Vincristine and Administration of Silibinin before the Chemical-Induced Injury. Table 8 shows the data on the proliferation of kidney cells damaged by vincristine under the constant influence of 20 and 40 µM silibinin hemisuccinate. At a concentration of 0.3 µM, vincristine inhibits growth by 60%. The administration of 20 and 40 µM silibinin hemisuccinate results in a reduction in growth inhibition to 56 and 51%, respectively.

	Discussion

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Because of its greater solubility, silibinin in the form of the hemisuccinate was used in the majority of experiments. This hemisuccinate is hydrolyzed rapidly and fully by the cells' esterases (Koch and Tscherny, 1983). Equivalent quantities of succinate fail to show any effect on the experimental data. Pure silibinin was used in the comparison of silibinin with silicristin, silidianin, and isosilibinin. The most notable finding is that silibinin increases the proliferation rate by up to 23% in Vero kidney cells, compared with the control cells. The concentration at which the efficacy is greatest is 40 µM silibinin hemisuccinate. Stimulation of comparable intensity was detected in the parallel measurements of DNA and protein biosynthesis with radioactive precursors. This stimulation decreases at higher concentrations, reaches the values of the controls at 60 µM, and changes into inhibition at concentrations in excess of 100 µM (Table 1). In the corresponding experiments with cells of the BSC-1 line, the results were essentially the same but the stimulatory effects were less pronounced (Table 1). Therefore, we focused our studies on Vero cells.

Sonnenbichler and Zetl (1986) found silibinin to have an identical effect on liver cells, for which RNA, DNA, and protein biosynthesis were measured. At concentrations in excess of 100 µM, similar inhibitions were observed in liver cells in vitro (Sonnenbichler et al., 1981). This substantiates the assumption that the same stimulation mechanism applies to kidney cells, as has been described in the case of liver cells in great detail (Machicao and Sonnenbichler, 1977). For liver it was shown that the DNA-dependent RNA-polymerase I is stimulated by interaction of silibinin to an enzyme effector binding site (Sonnenbichler and Zetl, 1988).

Repeated addition of stimulating silibinin hemisuccinate concentrations, staggered with respect to time, results in a reduction of the stimulatory effect. This confirms that the flavonolignan is not metabolized, but is accumulated by the kidney cells and thereby approaches the "inhibition range" of higher silibinin concentrations.

For the purpose of comparing the main components of the milk thistle extract, silibinin, isosilibinin, silicristin, and silidianin were also determined on Vero kidney cells with respect to the proliferation rates, the protein biosynthesis, and the LDH activities according to Stevens et al. (1986). LDH catalyzes the final stage of glycolysis, the reduction of pyruvate to lactic acid by NADH, one of the most important metabolic steps within cells. The enzyme plays an important role in the cytosol and reflects the vitality of the cells. Increases in protein synthesis also have an indirect effect on the LDH activity. It is noteworthy that the stimulatory effect of silibinin and silicristin on the LDH activities (Fig. 5) with increases of 18 and 14%, respectively, closely resembles the increases in proliferation of 16 and 14%, respectively (Table 2), and in protein biosynthesis of 16 and 11%, respectively, (Fig. 4). Isosilibinin and silidianin exhibit similar inhibitory effects in all three test systems.

It was also discovered in the case of liver cells (Andres, 1996) that silibinin and silicristin increase RNA and protein biosynthesis, whereas the remaining silidianin and isosilibinin exhibit little or no inhibitory effects. With regard to our research on the binding of silibinin to an effector site of the RNA-polymerase I (Zetl, 1985; Sonnenbichler and Zetl, 1988), it was revealed after molecular modeling studies in our laboratory, that indeed only silibinin and silicristin satisfy the requirements (suitable distance between functional groups, partial planarity, and resemblances between aromatic groups) for the binding to the receptor.

Because silibinin is capable of compensating for numerous forms of toxic damage in liver cells, it seemed appropriate to investigate whether flavonolignans can also prevent or reduce the detrimental effect of toxicants on kidney cells. This is of particular significance in the clinical application of cytostatics, but also for paracetamol, as its possible side effects include kidney damage. McMurtry et al. (1978) demonstrated that a high dose of paracetamol induces acute proximal tubular necroses in rats and in humans.

Cisplatin (cis-diamminedichloroplatinum) is a chemotherapeutic agent for the treatment of malignant tumors in humans (Prestayko et al.,1980), although its toxicity is high. Its dose limit can be attributed to its nephrotoxicity (Madias and Harrington, 1978, Erlanger and Cutler, 1992). As reported by Tay et al. (1988), DNA biosynthesis is inhibited by cisplatin. Various attempts have been made to reduce cisplatin-induced nephrotoxicity (Walker and Gale, 1981). These involved the use of various chemoprotectors, e.g., diethyl dithiocarbamate (Bodenner et al., 1986) and Ebselen (Baldew et al., 1992) for the purpose of lessening the cisplatin toxicity.

Vincristine, an alkaloid isolated from the evergreen Vinca rosea Linn., is also a cytostatic agent used in the treatment of chronic lymphatic leukemias, Hodgkin's disease, non-Hodgkin's lymphomas, neuroblastomas, and Wilms' tumors (Hahn et al., 1968; De Vita, 1981). Vincristine inhibits cells in the mitotic metaphase (Pouillart, 1993). Its biological activity is explained by its specific ability to bind to tubulin, thereby preventing polymerization of proteins to microtubules (Bowmann et al., 1988; Owellen et al., 1976). Carcinoma patients exhibited acute renal insufficiency after administration of vincristine (Jackson et al., 1984), e.g., increased excretion of renal enzymes was demonstrated by Dreller (1988) in patients with malignant melanomas during cytostatic perfusion therapy. According to Sharma et al. (1995), polyuria, enlarged kidneys, and renal insufficiency are the side effects of vincristine in the treatment of non-Hodgkin's lymphomas.

With these problems in mind, the effect of silibinin on damaged kidney cells was examined by administration of silibinin before and after the chemical-induced injury. In any case, the time kinetics of the selected parameters were measured, but for clear representation, only the values 72 h after silibinin addition are listed in the tables. The concentrations of paracetamol, cisplatin, and vincristine, capable of causing damage, were tested in preliminary experiments and subsequently selected so as to result in an approximate 20 to 30% inhibition of proliferation after 72 h.

The administration of silibinin 1 h after paracetamol intoxication shows distinct improvements in proliferation, protein biosynthesis, and LDH activities (Table 3). This effect becomes more pronounced if the relative improvements with respect to the damaged status are calculated: 16 to 26%. This also applies to previous administration of the flavonolignan 24 h before paracetamol-induced damage (Table 4), where relative improvements of 22 to 33% were reached.

With values of 26 to 28%, the effect of silibinin on Vero kidney cells previously damaged by cisplatin shows similar improvements in proliferation, protein biosynthesis, and LDH activity previously reduced by the toxicant (Table 5). In these experiments, it can be seen that the inhibitory effects of cisplatin on proliferation are considerably more pronounced than those on protein biosynthesis and LDH activities. As described, the cell volumes increase appreciably, possibly a compensation reaction. The disproportionately large growth inhibition can be attributed to the fact that, although RNA and protein biosynthesis are stimulated by silibinin, the inhibition of DNA synthesis (Tay et al., 1988) cannot be largely compensated by silibinin. The relative improvements, however, are similar for all three parameters. In the case of administration of silibinin before cisplatin damage, the relative stimulatory effects amount to 20 to 26%.

The effects of silibinin on parameters of cells damaged by vincristine are distinctly less marked. The relative improvements are 6 to 16% for subsequent and 10 to 22% for silibinin administration in advance.

With these experiments, it was possible to demonstrate that the flavonolignans isolated from the milk thistle, silibinin and silicristin, stimulate kidney cells in much the same way as they do liver cells. Considering the results of the experiments with kidney cells damaged by toxicants, the data warrant corresponding in vivo evaluations.