Alternative methods to safety studies in experimental animals: role in the risk assessment of chemicals under the new European Chemicals Legislation (REACH).

During the last two decades, substantial efforts have been made towards the development and international acceptance of alternative methods to safety studies using laboratory animals. In the EU, challenging timelines for phasing out of many standard tests using laboratory animals were established in the seventh Amending Directive 2003/15/EC to Cosmetics Directive 76/768/EEC. In continuation of this policy, the new European Chemicals Legislation (REACH) favours alternative methods to conventional in vivo testing, if validated and appropriate. Even alternative methods in the status of prevalidation or validation, but without scientific or regulatory acceptance may be used under certain conditions. Considerable progress in the establishment of alternative methods has been made in some fields, in particular with respect to methods predicting local toxic effects and genotoxicity. In more complex important fields of safety and risk assessment such as systemic single and repeated dose toxicity, toxicokinetics, sensitisation, reproductive toxicity and carcinogenicity, it is expected that the development and validation of in silico methods, testing batteries (in vitro and in silico) and tiered testing systems will have to overcome many scientific and regulatory obstacles which makes it extremely difficult to predict the outcome and the time needed. The main reasons are the complexity and limited knowledge of the biological processes involved on one hand and the long time frame until validation and regulatory acceptance of an alternative method on the other. New approaches in safety testing and evaluation using "Integrated Testing Strategies" (ITS) (including combinations of existing data, the use of chemical categories/grouping, in vitro tests and QSAR) that have not been validated or not gained wide acceptance in the scientific community and by regulatory authorities will need a thorough justification of their appropriateness for a given purpose. This requires the availability of knowledge and experience of experts in toxicology. The challenging deadlines for phasing out of in vivo tests in the Cosmetics Amending Directive 2003/15/EC appear unrealistic. Likewise, we expect that the application of validated alternative methods will only have a small or moderate impact on the reduction of in vivo tests under the regimen of REACH, provided that at least the same level of protection of human health as in the past is envisaged. As a consequence, under safety aspects, it appears wise to consider established in vivo tests to be indispensable as basic tools for hazard and risk assessment with respect to systemic single and repeated dose toxicity, sensitisation, carcinogenicity and reproductive toxicity, especially regarding quantitative aspects of risk assessment such as NOAELs, LOAELs and health-related limit values derived from them. Based on the overall evaluation in this review, the authors are of the opinion that in the short- and mid-term, the strategy of the development of alternative methods should be more directed towards the refinement or reduction of in vivo tests. The lessons learnt during these efforts will provide a substantial contribution towards the replacement initiatives in the long-term.

The Ginkgo biloba extract EGb761 reduces stress sensitivity, ROS accumulation and expression of catalase and glutathione S-transferase 4 in Caenorhabditis elegans.

The standardised extract EGb761 from the leaves of Ginkgo biloba is a popular herbal dietary supplement and it is used as a phytopharmacon for the therapy of diverse cerebral insufficiencies. The beneficial impact of EGb761 is believed to be conferred by diverse biological actions under physiological conditions as well as in response to stress. In this study we examined effects of EGb761 in the model organism Caenorhabditis elegans. EGb761 reduced the body size but did not affect the reproduction of C. elegans. In fluorescence-based assays performed in microtiter plates we demonstrated the protective action of EGb761 by the increase of resistance to thermal stress and the attenuation of ROS accumulation under conditions of thermal stress in single living worms. Under normal conditions the lifespan of the worms was extended by the EGb761 supporting the beneficial effects found under stress conditions. In a reporter gene approach using individual living worms the expression of the stress-inducible glutathione S-transferase 4 was shown to be reduced by EGb761 under physiological conditions as well as under oxidative stress. EGb761 also led to a decrease in transcription of the stress-inducible catalase genes. These results suggest that the beneficial impact of EGb791 on resistance to thermal stress and lifespan in C. elegans is at least partially due to its ability to relieve oxidative stress.

Prenylation enhances cytotoxicity of apigenin and liquiritigenin in rat H4IIE hepatoma and C6 glioma cells.

Antioxidative as well as cytotoxic effects of the prenylated flavonoids licoflavone C (8-prenylapigenin) and isobavachin (8-prenylliquiritigenin) were investigated in comparison to the corresponding non-prenylated flavonoids (apigenin, liquiritigenin) and vitexin (apigenin-C8-glucoside) using metabolically active H4IIE hepatoma and metabolically poorly active C6 glioma cells. None of the substances showed radical scavenging activities in the 2,2-diphenyl-1-picrylhydrazyl (DPPH)-assay nor were they effective in protection against H2O2-induced intracellular 2',7'-dichlorodihydrofluorescein (H2DCF) oxidation (fluorescent probe for oxidative stress) in H4IIE and C6 cells. When the intrinsic effects of the substances were investigated, licoflavone C and isobavachin exerted a pronounced toxicity in both H4IIE (IC50 values of 42+/-5 and 96+/-19 micromol/L) and C6 cells (IC50 values of 37+/-6 and 69+/-3 micromol/L) while the non-prenylated analogues as well as the glycosylated derivate vitexin showed almost no cytotoxic effect up to 250 micromol/L. In H4IIE cells the induction of apoptotic cell death by licoflavone C and icobavachin was detected as an activation of caspase 3/7 (6- and 3.3-fold, respectively). Based on these experiments we suggest that C8-prenylation of a flavonoid enhances the cytotoxicity inducing an apoptotic cell death in H4IIE cells without affecting antioxidative properties.

Resveratrol induces apoptotic cell death in rat H4IIE hepatoma cells but necrosis in C6 glioma cells.

Resveratrol (trans-3,5,4',-trihydroxystilbene) is assumed to possess cancer-preventive and cancer-therapeutic properties. The aim of this project was to analyze cellular effects of resveratrol in metabolically active H4IIE rat hepatoma cells in comparison to metabolically poorly active C6 rat glioma cells. Resveratrol is rapidly taken up by both cell types and acts as a potent intracellular antioxidant. On the other hand, resveratrol in higher concentrations is relatively toxic to both cell lines as measured by the neutral red accumulation assay. In H4IIE cells, resveratrol concentrations rapidly decline to very low levels during the first hours of incubation due to formation of resveratrol glucuronides. The first resveratrol effect found at 3h after the start of resveratrol treatment was the induction of mild DNA damage as detected by the comet assay. Cell death was caused via induction of apoptosis as detected by caspase activation, oligonucleosomal DNA fragmentation and formation of apoptotic nuclei. Following DNA damage, resveratrol led to an activation of caspases 2 and 8/10 at 6h and consequently of caspase 3 at 12h, but failed to activate caspase 9. In contrast to H4IIE cells, resveratrol is not metabolised in C6 glioma cells and accumulates to concentrations which are assumed to drive the cell into necrosis. This suggests that the mode of cell death caused by resveratrol and the usefulness of resveratrol for cancer prevention and treatment critically depends on the metabolic capacity of the tumor cell to be eradicated.

Pro-apoptotic effects of the flavonoid luteolin in rat H4IIE cells.

Polyphenols are ubiquitous substances in the diet. Their anti-oxidative, anti-inflammatory and anti-viral effects are of interest for human health, and polyphenols such as luteolin are used at high concentrations in food supplements. The aim of this project was to determine the intrinsic effects of luteolin in H4IIE rat hepatoma cells. Luteolin is relatively toxic, cell death was caused via induction of apoptosis as detected by DNA-ladder formation, by nuclear fragmentation and activation of apoptotic enzymes (caspase-2, -3/7, -9 and -8/10). Luteolin (250 microM, 24 h) increased the caspase-3/7 activity four-fold and the caspase-9 activity six-fold. In a time course experiment caspase-9 is activated after 6h, while caspase-2 and -3/7 are activated after 12 h. After 24 h, caspase-8/10 also displays activation. We found a concentration-dependent increase in malondialdehyde release suggesting a prooxidative effect of luteolin. Furthermore, we analysed DNA strand break formation by luteolin and found a distinct increase of DNA strand breaks after incubation for 3h with 100 microM luteolin, a concentration which induces oligonucleosomal DNA cleavage at 24h. In conclusion, the sequence of events is compatible with the assumption that luteolin triggers the mitochondrial pathway of apoptosis, probably by inducing DNA damage.

Oxidative stress in rat cortical neurons and astrocytes induced by paraquat in vitro.

Oxidative stress has been discussed as crucial mechanism of neuronal cell death in the adult brain. However, it was not clear until now whether neurons are more sensitive to oxidative stress than the other cells in the brain, e.g. astrocytes. Therefore both cell types were exposed to oxidative stress provoked by the redox-cycling compound paraquat. Cortical neurons were found to be more sensitive towards paraquat toxicity than astrocytes as shown by MTT and Neutral Red assay, two different cytotoxicity assays. Mitochondrial functions were determined by the mitochondrial membrane potential and intracellular ATP concentrations. Again cortical neurons were more severely impaired (by paraquat than astrocytes). The production of reactive oxygen species after paraquat exposure was much higher in cortical neurons than in astrocytes and correlated with a higher depletion of GSH (intracellular glutathion). Lipid peroxidation could be shown in astrocytes via the breakdown product malondialdehyde (MDA) whereas in cortical neurons 4-hydroxynonenal (4-HNE) was detected as this endpoint. If and how oxidative stress influences the antioxidant defense was determined via changes in the expression of antioxidant enzymes. Paraquat exposure lead to a 2-3 fold increase of catalase, MnSOD and CuZnSOD mRNA expression in astrocytes. In contrast to astrocytes, in cortical neurons catalase and MnSOD mRNA levels were only marginally elevated above 1.5-fold by treatment with paraquat. Expression levels of glutathione peroxidase (GPx) mRNA were the only one that were not changed in both cell types after paraquat exposure. It is concluded that the more marked increase in expression levels of antioxidant enzymes may render astrocytes more resistant to oxidative stress than neuronal cells.

Precision measurement of the spin-dependent asymmetry in the threshold region of 3He(e, e').

We present the first precision measurement of the spin-dependent asymmetry in the threshold region of 3He(e,e') at Q2 values of 0.1 and 0.2 (GeV/c)2. The agreement between the data and nonrelativistic Faddeev calculations which include both final-state interactions and meson-exchange current effects is very good at Q2 = 0.1 (GeV/c)2, while a small discrepancy at Q2 = 0.2 (GeV/c)2 is observed.

Changes in antioxidant enzyme expression in response to hydrogen peroxide in rat astroglial cells.

Oxidative stress has been causally linked to a variety of neurodegenerative diseases. To clarify the role of the antioxidant enzyme (AOE) system in oxidative brain damage primary cultures of rat astroglial cells were exposed to hydrogen peroxide (H2O2). Expression of AOEs and several parameters for cell viability and functionality were measured. In our experiments astrocytes responded to low concentrations of H2O2 exposure with a pronounced generation of ROS which ran parallel with induction of lipid peroxidation. This distinct oxidative stress was not reflected in cell viability or functionality parameters measured. Cytotoxicity, a decrease in glutathione content of astrocytes, and impairment of mitochondrial functions became obvious only for higher concentrations of H2O2. After H2O2 exposure catalase, manganese superoxide dismutase, and glutathione peroxidase expression levels were found to be increased, whereas copper/zinc superoxide dismutase mRNA expression was not affected. These data indicate that the AOE system of astrocytes can be directly regulated by oxidative stress and may thus contribute to protection of cells against oxidative insults.

The influence of oxidative stress on catalase and MnSOD gene transcription in astrocytes.

The brain is particularly vulnerable to oxygen free radicals, which have been implicated in the pathology of several neurological disorders. The antioxidant enzyme (AOE) system of the brain may play an important role in the protection against such oxidative stress. We investigated the influence of oxidative stress on the transcription of catalase and MnSOD mRNA. Primary rat astroglial cell cultures were treated either with hydrogen peroxide (H2O2), as a direct mediator of oxidative stress, or with the redox cycling compound paraquat. Both substances led to an increase of catalase and MnSOD mRNA levels. To further elucidate the mechanisms residing behind this increase, transfection experiments were performed. Transient transfection of primary astroglial cells with a reporter plasmid containing the upstream region of the catalase gene showed a decrease in reporter gene activity after exposure of transfected cells to either H2O2 or paraquat. In contrast, transfection experiments done with reporter plasmids for the MnSOD upstream region resulted in an increase of reporter gene activity after H2O2 as well as after paraquat treatment of transfected cells. These results indicate transcriptional regulation of MnSOD and post-transcriptional regulation of catalase gene expression after oxidative stress in primary rat astrocytes.

Transverse asymmetry AT' from the quasielastic 3He(e,e') process and the neutron magnetic form factor.

We have measured the transverse asymmetry A(T') in 3He(e,e(')) quasielastic scattering in Hall A at Jefferson Laboratory with high precision for Q2 values from 0.1 to 0.6 (GeV/c)(2). The neutron magnetic form factor G(n)(M) was extracted based on Faddeev calculations for Q2 = 0.1 and 0.2 (GeV/c)(2) with an experimental uncertainty of less than 2%.

Influence of Adriamycin and paraquat on antioxidant enzyme expression in primary rat hepatocytes.

The cytostatic Adriamycin and the herbicide paraquat form reactive oxygen species during enzymatic activation. Adriamycin, but not paraquat, is also able to intercalate into DNA and to interfere with DNA synthesis and transcription. We investigated the influence of both substances on antioxidant enzyme expression in primary rat hepatocytes. Treatment of hepatocytes with Adriamycin led to an increase in catalase and a decrease in MnSOD mRNA expression. In contrast, exposure of hepatocytes to paraquat resulted in an increase in both catalase and MnSOD message levels. CuZnSOD mRNA was not responsive to either treatment. Adriamycin almost completely inhibited RNA synthesis, but paraquat did not change either RNA or protein synthesis. Both substances induced lipid peroxidation as measured by the accumulation of malondialdehyde in the medium. These findings indicate that catalase and MnSOD are not regulated coordinately in hepatocytes and that ROS-producing agents can differentially influence expression of antioxidant enzymes depending on their capacity to inhibit transcription.

Effect of transforming growth factor-beta1 on cytochrome P450 expression: inhibition of CYP1 mRNA and protein expression in primary rat hepatocytes.

Primary hepatocytes are a widely used cell model to analyse the expression and regulation of hepatic cytochrome P450 (CYP) isoenzymes. Transforming growth factor-beta1 (TGF-beta1) was previously shown to inhibit constitutive and induced CYP1 expression in human cell lines and primary hepatocytes but not in rat cells. In the present study we examined the effect of TGF-beta1 on constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced expression of CYP1 isoenzymes in primary rat hepatocytes in order to address the species-specificity of CYP1 down-regulation by TGF-beta1. The results show an inhibition of TCDD-induced CYP1-related 7-ethoxyresorufin-O-deethylase (EROD), 7-methoxyresorufin-O-demethylase (MROD) activities and mRNA expression (determined by reverse transcriptase polymerase chain reaction, RT-PCR) by 100 pM TGF-beta1 in cells co-treated for 24 h with 1 nM TCDD. However, while TGF-beta1 also down-regulated constitutive EROD and MROD activities as well as CYP1A2 protein expression, it did not change the constitutive mRNA expression of CYP1 isoenzymes. The down-regulation seemed to be specific for CYP1 isoenzymes since constitutive expression of other CYP isoenzymes was unaffected concerning protein levels, as determined by Western blot for CYP2B1/2 and CYP3A1, as well as mRNA levels, as determined by RT-PCR for CYP2B1/2, CYP2E1 and CYP3A1. Thus, TGF-beta1 not only inhibits CYP1 expression in humans but also in rats, indicating that regulation of CYP1 expression in these two species is similar.

Regulation of prostaglandin endoperoxide H synthase-2 induction by dioxin in rat hepatocytes: possible c-Src-mediated pathway.

The tumor promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to increase the expression of prostaglandin endoperoxide H synthase (PGHS)-2. This study focused on the regulatory mechanism of TCDD-mediated transcriptional activation of PGHS-2. Treatment of rat hepatocytes with TCDD led to a dose-dependent induction of PGHS-2 mRNA levels associated with an increased synthesis of prostaglandin E(2), whereas expression of PGHS-1 was not affected. In vitro experiments with c-Src inhibitors, such as herbimycin A and geldanamycin, and in vivo studies with c-Src-deficient mice indicated that up-regulation of PGHS-2 but not the cytochrome P450 gene CYP1A1 by TCDD is mediated via a c-Src-dependent pathway. Transient transfection studies with different reporter constructs of the murine PGHS-2 promoter mutated in the xenobiotic-responsive element (XRE) or CCAAT/enhancer binding protein (C/EBP) element revealed that a C/EBP-binding site is an important regulatory cis-acting factor for trans-activation of the PGHS-2 gene by TCDD. Consistent with transfection studies, gel mobility shift assays showed that TCDD led to an enhanced DNA-binding activity of C/EBP beta transcription factor. The experimental data presented in this article reveal a XRE-independent and c-Src-mediated activation of the PGHS-2 gene by TCDD through the C/EBP response element located in its promoter region.

Influence of tumor necrosis factor-alpha and silibin on the cytotoxic action of alpha-amanitin in rat hepatocyte culture.

Tumor necrosis factor-alpha is assumed to play a role in toxic liver damage. We examined whether exogenous tumor necrosis factor-alpha must be present for alpha-amanitin cytotoxicity in rat hepatocyte culture. alpha-Amanitin at a concentration of 0.1 microM, which is close to that found in intoxicated patients, inhibits RNA and protein synthesis within 12 h but cytotoxicity only occurs after a latency period and is pronounced at 36 h after the start of treatment. Tumor necrosis factor-alpha is not indispensable for the development of cytotoxicity but aggravates it and leads to a time shift towards earlier times. Lipid peroxidation is low with alpha-amanitin alone even at 36 h but markedly increased by cotreatment with tumor necrosis factor-alpha. The antioxidant silibin prevents the effect of tumor necrosis factor-alpha, indicating an involvement of reactive oxygen species. alpha-Amanitin alone does not increase but dose-dependently inhibits the expression of the antioxidant enzyme manganous superoxide dismutase and decreases the inducing effect of TNF-alpha on the expression of this enzyme. The gene expression of endogenous tumor necrosis factor-alpha in the hepatocytes is not increased but rather inhibited by alpha-amanitin treatment. The results suggest that alpha-amanitin causes delayed cytotoxicity following rapid inhibition of RNA and protein synthesis and that tumor necrosis factor-alpha shortens the latency period and aggravates the cytotoxicity by a mechanism which may involve reactive oxygen species.

Impairment of TNF-alpha expression and secretion in primary rat liver cell cultures by acetaminophen treatment.

Tumor necrosis factor-alpha (TNF-alpha) is assumed to act as a mediator in toxic liver injury, aggravating the primary damage to the parenchymal liver cell, but also stimulating liver regeneration. Reports on the effect of acetaminophen in vivo on TNF-alpha transcript concentrations and serum TNF-alpha concentrations, under different experimental, or clinical conditions have yielded controversial results. We used primary rat hepatocyte and Kupffer cell cultures to test the direct action of subtoxic and toxic concentrations of acetaminophen on TNF-alpha expression and release. We observed a dose-dependent decrease of TNF-alpha mRNA in the hepatocytes, and of TNF-alpha release into the medium of hepatocyte cultures. The data also indicate an impairment of TNF-alpha release in Kupffer cell cultures after treatment with nontoxic, as well as with toxic, acetaminophen concentrations. The results suggest that inhibition of TNF-alpha expression and release in the liver is a consequence of acetaminophen exposure. It is at present unknown how this effect modulates the course of acetaminophen intoxication.