Claus-Peter Siegers

The influence of fasting on the susceptibility of mice to hepatotoxic injury.

The hepatotoxic effects of eight compounds as determined by serum activities of glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), and sorbitol dehydrogenase (SDH) were investigated in normally fed mice on the one hand and 24-hr-fasted mice on the other. Fasting strongly enhanced serum-enzyme elevations induced by carbon tetrachloride, paracetamol, thioacetamide, and bromobenzene. The hepatotoxic effects of phalloidin and allyl alcohol were only moderately increased by fasting and those of α-amanitin and praseodymium not at all. The fasting-induced aggravation of CCl4 hepatotoxicity (evidenced also by histological findings) appeared already after 12 hr and was maximal after 24 hr of food deprivation. Fasting for 24 hr decreased the liver weight by 29%, hepatic glycogen by 93%, and hepatic glutathione (GSH) by 50% but increased liver triglycerides by 162%. Aniline hydroxylase and aminopyrine N-demethylase activities were higher in the liver homogenate supernatants from fasted than from fed mice but microsomal protein content as well as microsomal NADPH-cytochrome c-reductase activity remained unchanged and microsomal cytochrome P-450 content even decreased upon fasting. Fasting did not influence the in vitro irreversible binding of 14CCl4 and [3H]paracetamol to hepatic microsomal proteins nor the in vivo binding to hepatic proteins of [3H]paracetamol. It enhanced, however, the total concentration of 14CCl4 in the liver by 30% and produced a trend toward higher values in the extent of 14CCl4 bound in vivo to hepatic protein. Spontaneous and CCl4-induced lipid peroxidation were the same in hepatic microsomes from fed and fasted mice. No unique explanation can be made for the increased susceptibility of mouse liver to toxic injury induced by fasting. Several factors must be considered: depletion of both hepatic glycogen and glutathione as well as hepatic lipid accumulation. Overnight fasting of mice (and of other small animals presumably too) may change the results of toxicological experiments to an unpredictable degree and should thus be avoided.

In vitro evaluation of the cytotoxic potential of alkylphenols from Ginkgo biloba L.

Extracts from the leaves of Ginkgo biloba L. belong to the most widely used phytopharmaceuticals. In crude Ginkgo extracts, ginkgolic acids (GA) and related alkylphenols (e.g. cardanols and cardols) have been recognized as hazardous compounds with suspected cytotoxic, allergenic, mutagenic and carcinogenic properties. To further assess the cytotoxic potential of GA, their effect on the human keratinocyte cell line HaCaT and the rhesus monkey kidney tubular epithelial cell line LLC-MK(2) was investigated. The action of a defined mixture of GA on cell growth, viability and integrity was evaluated by the neutral red uptake assay as well as the release of lactate dehydrogenase (LDH) and acid phosphatase (ACP). Cell morphology was examined by electron microscopy. For comparison, the effect of the standardized Ginkgo extract EGb 761, which contains less than 5 ppm GA, was also investigated. Following incubation of cells with EGb 761, neutral red uptake was half-maximally inhibited at concentrations of 900 mg/l (HaCaT) and 1480 mg/ml (LLC-MK(2)). The corresponding IC(50)-values for the mixture of GA ranged between 22 mg/l (HaCaT) and 4.6 mg/l (LLC-MK(2)), respectively. In parallel to the inhibition of neutral red uptake, a concentration dependent release of LDH was observed when cells were incubated in the presence of GA (1-100 mg/l). In contrast, even at a concentration of 1800 mg/l EGb 761 did not cause release of LDH above controls. Since GA interacted with the assay for ACP, no index of lysosomal damage could be established by this method. Incubation of HaCaT cells with GA for 18 h increased the proportion of apoptotic cells from about 6% (control) to nearly 80% at concentrations of >or=30 mg/l. Electron microscopic analysis of HaCaT cells revealed a drug induced formation of myelinosomes possibly due to the inhibition of lysosomal enzymes, while morphological evaluation of LLC-MK(2) cells indicated that the cytotoxic activity of GA in these cells is primarily mediated by transformation of mitochondria, which is probably induced by uncoupling of oxidative phosphorylation.

The effects of garlic preparations against human tumor cell proliferation

Epidemiological studies in China provide reason to suspect that a rich garlic content in the diet might reduce the proliferation of tumors in humans. We conducted experiments on human tumor cell lines and determined the influence of a garlic powder preparation, a garlic extract (reported as 8-10% L(+)-alliin enriched), and a combination thereof, on cellular proliferation in cell cultures, employing the widely used indirect neutral red procedure. Garlic powder failed to inhibit the growth of human hepatoma HepG2 or human colorectal carcinoma Caco2 cells at concentrations of up to 1000 micrograms/ml. Garlic extract, in which the alliin content was highly enriched was also unable to inhibit the growth of these cells. However, when the garlic extract was supplemented with garlic powder (to 10% final concentration) there was a concentration-dependent clear inhibition of tumor cell growth (IC50 values of 330 micrograms/ml for HepG2 and 480 micrograms/ml for Caco-2 cells). The growth of the human lymphatic leukemia cell line CCRF CEM was significantly inhibited in a dose-dependent manner by both garlic powder and garlic extract at concentrations as low as 30 micrograms/ml. However, no potentiation of this effect occurred upon mixing of the two preparations. Our results suggest that the antiproliferative effects of garlic may be due to breakdown products of alliin, such as allicin or polysulfides, rather than alliin itself, since the addition of an alliinase system (garlic powder) to an alliin enriched preparation without alliinase (garlic extract) potentiated the effects observed with the two preparations alone.

The role of iron in the paracetamol- and CCl4-induced lipid peroxidation and hepatotoxicity

Treatment of non-induced or phenobarbital-induced, glutathione-depleted mice with 400 mg/kg paracetamol led to a marked ethane exhalation as an index of in vivo lipid peroxidation (LPO) and to a significant elevation of liver-specific serum enzyme activities. Similar effects were seen with rats treated with 0.5 ml/kg CCl4. Pretreatment with the iron-chelating agent desferrioxamine (DFO) clearly suppressed lipid peroxidation in all cases, but inhibited only the CCl4-induced hepatotoxicity. Treatment of mice with desferrioxamine alone showed no hepatotoxicity at all, nor did it influence liver GSH-levels. In addition, DFO had no effect on hepatic microsomal enzyme activities responsible for the bioactivation of both paracetamol and CCl4. These findings are consistent with the theories which indicate that lipid peroxidation requires the presence of Fe2+-ions, regardless of the initiating agent, and that LPO is involved in CCl4-toxicity, but most probably not in paracetamol-induced liver damage. Furthermore, Fe2+-ions might play a role as mediators of CCl4-hepatotoxicity.

Toxicity of kava pyrones, drug safety and precautions: a case study

Kava pyrones have been sold in Germany as OTC anxiolytics until June 2002, when all preparations with a kava pyrone content of more than 10(-4) of a homeopathic stock solution were withdrawn. Other countries in which kava pyrones have been used as anxiolytics, namely GB and the USA, have not followed suit. Kava pyrone anxiolytics have been positively reviewed by the Cochrane Collaboration; also newer German clinical studies have indicated pharmacological anxiolysis at the recommended doses. To use the first choice of treatment, psychotherapy, for all uncomplicated cases of pathological fear does not appear to be realistic. Current data about kava pyrone toxicity are unclear. Judging from the few well documented cases of kava pyrone hepatotoxicity (appr. 2 out of 36) in Germany and Switzerland, an immunologically mediated idiosyncratic mechanism appears to be most likely, especially at higher doses, whereas a direct toxic mechanism is much less likely. No direct results are available for the incidence of kava pyrone-related adverse drug effects. From spontaneously reported cases the incidences of adverse drug reactions cannot be obtained, a rough estimation indicates the incidence of hepatotoxicity to be comparable to those of benzodiazepines. Taken together, the withdrawal of kava pyrone-based anxiolytics appears to be an ill founded over-reaction given the lack of superior therapeutic alternatives. Neither the case evaluations presented by the BfArM (Bundesamt für Arzneimittel und Medizinprodukte = Federal Office for Drugs and Medical Products) nor the complete rejection of proof for therapeutic efficacy of kava pyrone anxiolytics are scientifically well founded.

Increased carbon tetrachloride hepatotoxicity after low-level ethanol consumption

Male rats provided with a 5 or 15% (v/v) ethanol solution as the sole source of fluid consumed ethanol at a rate of 11.4 or 24.9% of total calories (4.2 or 8.3 g/kg daily). After ethanol consumption lasting 1, 2 and 3 weeks the hepatotoxicity of CCl4 (0.1 ml/kg i.p.) was elevated by determination of serum activities of glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase ( GPT), sorbitol dehydrogenase (SDH) and histological investigations. Carbon tetrachloride (CCl4)-induced liver damage was significantly greater in rats provided with ethanol than in the tap-water consuming controls. This potentiation of CCl4 hepatotoxicicty was fully developed already after a 1-week exposition to ethanol and was greater in the 15% than in the 5% ethanol group. Ethanol alone did not influence serum enzyme activities but increased microsomal aniline hydroxylation. There was, however, no clear-cut parallelism between potentiation of CCl4 hepatotoxicity and activation of aniline hydroxylation.

Influence of cadmium chloride, mercuric chloride, and sodium vanadate on the glutathione-conjugating enzyme system in liver, kidney, and brain of mice.

Sublethal doses of CdCl2 (3 mg/kg iv), HgCl2 (2 mg/kg iv), or NaVO3 (6 mg/kg iv) did not alter the content of reduced glutathione (GSH) in the livers of mice during the 24-h observation period. In the kidneys, a tendency to increased GSH content was seen, especially after HgCl2 treatment; in lung and brain the GSH levels were significantly lowered upon the treatment with all three metals. The activities of GSH S-transferase toward an aryl substrate (CDNB; 1-chloro-2,4-dinitrobenzene) was enhanced in all tissues by the administration of HgCl2 greater than NaVO3 greater than CdCl2. The activity of GSH S-transferase toward an epoxide substrate [1,2-epoxy-3-(p-nitrophenoxy)propane was only measurable in the livers and was inhibited 1 and 2 h after the administration of HgCl2 and NaVO3. It is concluded that sublethal doses of CdCl2, HgCl2, or NaVO3 do not impair the GSH concentration and GSH-conjugating enzyme activities toward the aryl substrate in different target organs of their toxicity, which is in contrast to results obtained in vitro.

Role of flavonoids in controlling the phototoxicity of Hypericum perforatumextracts

Hypericum perforatum extracts are used mainly as oral antidepressants. Depending on source the extracts contain various amounts of phenylpropanes, flavonol derivates, biflavones, proathocyanidines, xanthones, phloroglucinoles, some amino acids, naphtodianthrones (hypericines) and essential oil constituents. The therapeutic use of Hypericum perforatum extracts however is limited by their phototoxic potential. It was the aim of the present study to investigate the phototoxic potential of 3 Hypericum perforatum extracts from different sources as well as some of its main constituents. In order to systematically study the phototoxic potential we established a modified neutral red assay utilizing an immortalized human keratinocyte cell line (HaCaT cells) as substrate and UVA irradiation. This modified neutral red assay was found to be a simple and reliable method for detecting phototoxic effects of reference agents and plant extracts. The validity of this method was demonstrated with known phototoxic compounds like chloropromazine and psoralenes like 5-MOP. Hypericum perforatum extracts demonstrated cytotoxicity and photocytotoxicity in a dose and UVA-dose dependent manner. Hypericine itself also evoked severe phototoxic effects and was thus identified as the main phototoxic constituent. Among the tested flavonoids quercitrin was found to be cytotoxic, while rutin unexpectedly demonstrated phototoxicity whereas quercitrin was effective to control the phototoxic activity of Hypericum perforatum extracts.

Cytotoxicity of heavy metals in the human small intestinal epithelial cell line I‐407: The role of glutathione

Cytotoxicities of metal salts were determined in the intestinal epithelial cell line I-407 in microwell culture plates over 48 h using the widely utilized and accepted neutral red uptake procedure. Rank order cytotoxicities induced by the metal salts (in terms of LC50 values) were found to be HgCl2 (32 microM) > CdCl2 (53 microM) > CuCl2 (156 microM) > T12SO4 (377 microM) > Pb(NO3)2 (1.99 mM). Combined administration of the two most toxic metals at their LC50s showed that their toxicities were not additive or synergistic. The role of glutathione in determining toxicity induced by the metal salts in these cells was assessed by inhibition of its synthesis. Buthionine sulfoximine pretreatment at 1 mM, which was not toxic to the cells, caused sustained reduction in cellular glutathione content (to 13.8% after 48 h) and increased toxicities induced by HgCl2 (5.7-fold) and CuCl2 (1.44-fold) as shown by reductions in the LC50 values. Toxicity induced by the other metals remained unaffected. Administration of glutathione with either HgCl2 or CdCl2 did not protect the cells against their toxicity, and in the case of cadmium its toxicity was exacerbated. N-Acetylcysteine diminished toxicity induced by mercury but not cadmium.

Glutathione Depletion by Phorone Organ Specificity and Effect on Hepatic Microsomal Mixed-Function Oxidase System

Phorone (diisopropylidene acetone) led to a strong depletion of cellular glutathione in liver, kidney and heart but not in lung or brain upon administration to mice or rats. Its efficacy in lowering hepatic glutathione levels was comparable to that of diethylmaleate. Unlike this agent, however, phorone did not affect the microsomal mixed-function oxidase system at all. Our findings favor the use of phorone when studying the effect of a decreased glutathione content on detoxication, bioactivation or drug metabolism mechanisms, as it merely interacts with glutathione itself.