Rolf Schulte-Hermann

Review of the toxicology of chlorpyrifos with an emphasis on human exposure and neurodevelopment

This review examines the large body of toxicological and epidemiological information on human exposures to chlorpyrifos, with an emphasis on the controversial potential for chlorpyrifos to induce neurodevelopmental effects at low doses. The results of this review demonstrate that the use of urinary 3,5,6-trichlorpyridinol (TCPy), a metabolite of chlorpyrifos as a biomarker of nonoccupational exposure is problematic and may overestimate nonoccupational exposures to chlorpyrifos by 10-to 20-fold because of the widespread presence of both TCPy and chlorpyrifos-methyl in the food supply. Current “background” (nonoccupational) levels of exposure to chlorpyrifos are several orders of magnitude lower than those required to inhibit plasma cholinesterase activity, which is a more sensitive target than nervous system cholinesterase. However, several in vitro studies have identified putative neurodevelopmental mechanisms that are altered at concentrations of chlorpyrifos below those that inhibit cholinesterases. Although one human cohort study reported an association between maternal and cord blood chlorpyrifos levels and several measures of neurodevelopment, two other cohort studies that utilized urinary TCPy as a surrogate for chlorpyrifos exposure did not demonstrate an association. Although the weight of the scientific evidence demonstrates that current levels of chlorpyrifos exposure will not have any adverse effects on neurodevelopment that might result from inhibition of nervous system cholinesterases, several recent studies propose alternative mechanisms. Thus, further in vivo investigation on neurodevelopment in an appropriate animal model is needed; additional epidemiological studies may be warranted if a suitable, chlorpyrifos-exposed cohort can be identified and more rigorous measures of exposure are utilized.

Molecular aspects of epithelial cell plasticity: implications for local tumor invasion and metastasis

Carcinomas arising from epithelial cells represent the most prevalent malignancies in humans, and metastasis is the major cause for the death of carcinoma patients. The breakdown of epithelial cell homeostasis leading to aggressive cancer progression has been correlated with the loss of epithelial characteristics and the acquisition of a migratory phenotype. This phenomenon, referred to as epithelial to mesenchymal transition (EMT), is considered as a crucial event in late stage tumorigenesis. Here we summarize the multitude of EMT models derived from different tissues, and review the diversity of molecular mechanisms contributing to the plasticity of epithelial cells. In particular, the synergism between activation of Ras, provided by the aberrant stimulation of receptor tyrosine kinases, and transforming growth factor (TGF)-beta signaling plays a pivotal role in inducing EMT of various epithelial cell types. Cytokines such as TGF-beta and extracellular matrix molecules are thought to fundamentally contribute to the microenvironmental interaction between stromal and malignant cells, and provide the basis for a broad repertoire of epithelial differentiation. Investigations of EMT tumor models, which represent in vitro correlates to local invasion and metastasis in vivo, facilitate the identification of diagnostic markers for a more accurate and faithful clinical and pathological assessment of epithelial tumors. In addition, the analysis of molecular mechanisms involved in EMT might yield novel therapeutic targets for the specific treatment of aggressive carcinomas.

Green tea extract and (–)-epigallocatechin-3-gallate, the major tea catechin, exert oxidant but lack antioxidant activities

Green tea is the most widely consumed beverage. It has attained high reputation as a health‐promoting dietary component ascribed to the antioxidant activity of (−)‐epigallocatechin‐3‐gallate (EGCG), its main polyphenolic constituent. Evidence is increasing that tea constituents can be cell damaging and pro‐oxidant themselves. These effects were suggested to be due to spontaneous H2O2 generation by polyphenols in solution. In the present study, we investigated the oxidant and antioxidant properties of green tea extracts (GTE) and of EGCG by means of the rodent macrophage‐like RAW 264.7 and human promyelocytic leukemic HL60 cell lines. The results obtained show that both under cell‐free conditions and in the presence of cells the oxidant activities of GTE and EGCG exceeded those of spontaneously generated H2O2 (FOX assay). Increase of intracellular oxidative stress was indicated by 2′,7′‐dichlorofluorescin probing, and the enhanced genotoxicity was demonstrated by the alkaline comet assay and by the micronucleus assay (cytokinesis block). Time‐ and dose‐dependent induction of cell death was monitored by trypan blue exclusion, MTT assay, and Hoechst staining. Furthermore, in our systems in vitro, EGCG neither directly scavenges H2O2 nor mediates other antioxidant activities but rather increased H2O2‐induced oxidative stress and DNA damage. In conclusion, our data suggest that detailed mechanistic studies on the effects of GTE and EGCG should be performed in vivo before excessive intake and/or topical application of green tea products can be recommended to healthy and/or diseased persons.

Transforming growth factor-beta 1 as a signal for induction of cell death by apoptosis.

Cell death by apoptosis is a major determinant of growth of normal tissues and tumours. The present study aimed to elucidate signal factors involved in its regulation. Epithelial cells in control liver, during regression of cyproterone acetate induced liver hyperplasia, in liver (pre)neoplasia and in uterus undergoing apoptosis in vivo show immunostaining for transforming growth factor beta 1 (TGF-beta 1) as detected by anti-pre(266-278) TGF-beta 1 antibodies. Positive immunostaining is also seen in a few intact cells of hyperplastic, regressing liver apparently preparing for apoptosis, but is virtually not found in hepatocytes of normal or growing liver nor in cells undergoing death by necrosis. Recombinant latency associated protein (rLAP, dimer of the pro-region non-covalently associated with the mature region) complex and mature TGF-beta 1 induce apoptosis in isolated hepatocytes cultured in vitro. These findings suggest an involvement of TGF-beta 1 in the induction of apoptosis in certain epithelia in vivo.

The Fas-induced apoptosis analyzed by high throughput proteome analysis.

The fate of cytosolic proteins was studied during Fas-induced cell death of Jurkat T-lymphocytes by proteome analysis. Among 1000 spots resolved in two-dimensional gels, comparison of control versus apoptotic cells revealed that the signal intensity of 19 spots decreased or even disappeared, whereas 38 novel spots emerged. These proteins were further analyzed with respect tode novo protein synthesis, phosphorylation status, and intracellular localization by metabolic labeling and analysis of subcellular protein fractions in combination with two-dimensional Western blots and mass spectrometry analysis of tryptic digests. We found that e.g. hsp27, hsp70B, calmodulin, and H-ras synthesis was induced upon Fas signaling. 34 proteins were affected by dephosphorylation (e.g. endoplasmin) and phosphorylation (e.g. hsc70, hsp57, and hsp90). Nuclear annexin IV translocated to the cytosol, whereas decreasing cytosolic TCP-1α became detectable in the nucleus. In addition, degradation of 12 proteins was observed; among them myosin heavy chain was identified as a novel caspase target. Fas-induced proteome alterations were compared with those of other cell death inducers, indicating specific physiological characteristics of different cell death mechanisms, consequent to as well as independent of caspase activation. Characteristic proteome alterations of apoptotic cells at early time points were found reminiscent of those of malignant cells in vivo.

Khat (Catha edulis) consumption causes genotoxic effects in humans

We used the micronucleus (MN) test to determine the genetic damage caused by khat, a widely consumed psychostimulant plant, in exfoliated cells of volunteers who chewed the drug on a regular basis. In the first study in which we compared the frequency of MN in buccal and bladder mucosa cells in 20 khat consumers (10–160 g/day) and 10 controls, a pronounced (8‐fold) increase in micronucleated buccal mucosa cells was seen among khat consumers; khat consumption did not lead to a detectable elevation of micronucleated bladder mucosa cells. Among heavy khat chewers, 81% of the MN had a centromere signal indicating that khat is aneuploidogenic. To investigate the effect of simultaneous consumption of tobacco and alcoholic beverages, we compared the MN frequency in buccal cells of 25 khat consumers (20–85 g/day) who smoked cigarettes (15–60/day) and drank alcoholic beverages (15–80 g of pure ethanol/day) with a control group (control group I) of 25 individuals matched for age, body weight, tobacco and alcohol consumption and with another control group of 25 individuals (control group II) not consuming any of the drugs. The frequency of buccal mucosa cells with MN was higher in control group I than in group II and the effect of khat, tobacco and alcohol was found to be additive. A time‐kinetics study on khat‐induced MN showed that the highest frequency of MN was observed during the fourth week after consumption. In light of the large body of evidence on the close association between genetic damage and cancer, these results suggest that khat consumption, especially when accompanied by alcohol and tobacco consumption, might be a potential cause of oral malignancy.

Cell death and autophagy: Cytokines, drugs, and nutritional factors

Cells may use multiple pathways to commit suicide. In certain contexts, dying cells generate large amounts of autophagic vacuoles and clear large proportions of their cytoplasm, before they finally die, as exemplified by the treatment of human mammary carcinoma cells with the anti-estrogen tamoxifen (TAM, < or = 1 microM). Protein analysis during autophagic cell death revealed distinct proteins of the nuclear fraction including GST-pi and some proteasomal subunit constituents to be affected during autophagic cell death. Depending on the functional status of caspase-3, MCF-7 cells may switch between autophagic and apoptotic features of cell death [Fazi, B., Bursch, W., Fimia, G.M., Nardacci R., Piacentini, M., Di Sano, F., Piredda, L., 2008. Fenretinide induces autophagic cell death in caspase-defective breast cancer cells. Autophagy 4(4), 435-441]. Furthermore, the self-destruction of MCF-7 cells was found to be completed by phagocytosis of cell residues [Petrovski, G., Zahuczky, G., Katona, K., Vereb, G., Martinet, W., Nemes, Z., Bursch, W., Fésüs, L., 2007. Clearance of dying autophagic cells of different origin by professional and non-professional phagocytes. Cell Death Diff. 14 (6), 1117-1128]. Autophagy also constitutes a cells strategy of defense upon cell damage by eliminating damaged bulk proteins/organelles. This biological condition may be exemplified by the treatment of MCF-7 cells with a necrogenic TAM-dose (10 microM), resulting in the lysis of almost all cells within 24h. However, a transient (1h) challenge of MCF-7 cells with the same dose allowed the recovery of cells involving autophagy. Enrichment of chaperones in the insoluble cytoplasmic protein fraction indicated the formation of aggresomes, a potential trigger for autophagy. In a further experimental model HL60 cells were treated with TAM, causing dose-dependent distinct responses: 1-5 microM TAM, autophagy predominant; 7-9 microM, apoptosis predominant; 15 microM, necrosis. These phenomena might be attributed to the degree of cell damage caused by tamoxifen, either by generating ROS, increasing membrane fluidity or forming DNA-adducts. Finally, autophagy constitutes a cells major adaptive (survival) strategy in response to metabolic challenges such as glucose or amino acid deprivation, or starvation in general. Notably, the role of autophagy appears not to be restricted to nutrient recycling in order to maintain energy supply of cells and to adapt cell(organ) size to given physiological needs. For instance, using a newly established hepatoma cell line HCC-1.2, amino acid and glucose deprivation revealed a pro-apoptotic activity, additive to TGF-beta1. The pro-apoptotic action of glucose deprivation was antagonized by 2-deoxyglucose, possibly by stabilizing the mitochondrial membrane involving the action of hexokinase II. These observations suggest that signaling cascades steering autophagy appear to provide links to those regulating cell number. Taken together, our data exemplify that a given cell may flexibly respond to type and degree of (micro)environmental changes or cell death stimuli; a cells response may shift gradually from the elimination of damaged proteins by autophagy and the recovery to autophagic or apoptotic pathways of cell death, the failure of which eventually may result in necrosis.

Effect of Staining Procedures on the Results of Micronucleus Assays with Exfoliated Oral Mucosa Cells

Micronuclei in exfoliated epithelial cells are widely used as biomarkers of cancer risk in humans. To elucidate the effect of different staining procedures on the outcome of such investigation, we conducted a study in which the micronuclei frequencies in oral mucosa cells of heavy smokers (n = 20) and nonsmokers (n = 10) were evaluated with nonspecific (Giemsa, May-Grünwald-Giemsa) and DNA-specific (4′,6-diamidino-2-phenylindole, Feulgen, acridine orange) stains, whereas with Giemsa-based stains, the frequencies of micronuclei in smokers were significantly (4- to 5-fold) higher in the smokers group, no significant increase was observed with any of the DNA-specific stains. Furthermore, the evaluation of cells of the two study groups with Feulgen stain showed that oral mucosa cells from smokers had significantly increased levels of nuclear anomalies other than micronuclei. These anomalies are consequences of cell injury found in epithelial cells and are paralleled by formation of keratin bodies in the cytoplasm that resemble micronuclei. Correlation analyses showed that micronuclei frequencies scored in Giemsa-stained slides correlated significantly with karyorrhexis, karyolysis, condensed chromatin, and binucleates, whereas no such correlations were found with DNA-specific stains. These findings indicate that nuclear anomalies (and possibly keratin bodies) may be misinterpreted as micronuclei with nonspecific DNA stains and lead to false-positive results in studies with cells of epithelial origin. Furthermore, our results show that exposure of oral mucosa cells to genotoxic carcinogens contained in tobacco smoke does not lead to induction of micronuclei in these cells. (Cancer Epidemiol Biomarkers Prev 2006;15(10):1835–40)

The coffee components kahweol and cafestol induce γ-glutamylcysteine synthetase, the rate limiting enzyme of chemoprotective glutathione synthesis, in several organs of the rat

Abstract. The coffee components kahweol and cafestol (K/C) were reported to be protective against mutagenic damage by heterocylic amines and aflatoxin B1 in the rat, while in humans the consumption of coffee with a high K/C content was associated with a lower rate of colon tumors. An important mechanism of this antimutagenic effect appears to be the potential of K/C to induce glutathione-S-transferase (GST) and to enhance hepatic levels of glutathione (GSH), the co-factor of GST, which is independently involved in further protective mechanisms. In the present study, we investigated mechanisms and organ specificities (liver, kidney, lung, colon) of the K/C effect on GSH levels, and particularly the role of γ-glutamylcysteine synthetase (GCS), the rate limiting enzyme of GSH synthesis. Chows containing one of four concentrations of either a 1:1 mixture of K/C (0.012–0.122%) or of cafestol alone (0.006–0.061%) were fed to male F344 rats for 10 days. In the K/C-treated livers, a dose-dependent increase of up to 2.4-fold in the activity of GCS was observed, being statistically significant even at the lowest dose, and associated with an increase in GSH of up to three-fold. Notably, the highest dose doubled the hepatic mRNAs of the heavy and light subunits of GCS, suggesting enhanced transcription. In the extrahepatic organs, GCS activity and GSH levels were increased as well, although more moderately than in the liver. Since enhancement of GCS had also been observed as a consequence of oxidative stress, the possibility of such an involvement in the actions of K/C was examined by determining hepatic thiobarbituric acid reactive substances and the ratio of oxidized and reduced GSH. However, no evidence of oxidative stress was detected. In summary, K/C increased GSH levels apparently through the induction of the rate limiting enzyme of GSH synthesis, which may be a key factor in the chemopreventive potential of coffee components.

Search for Compounds That Inhibit the Genotoxic and Carcinogenic Effects of Heterocyclic Aromatic Amines

Over the last 30 years approximately 160 reports have been published on dietary compounds that protect from the mutagenic and carcinogenic effects of heterocyclic aromatic amines (HAAs). In the first section of this review, the current state of knowledge is briefly summarized. Based on the evaluation of the available data, various protective mechanisms are described, and the use of different methodologies for the detection of protective effects is critically discussed. In most antimutagenicity studies (>70%) bacterial indicators (predominantly Salmonella strain TA98) were used, and about 600 individual compounds and complex mixtures have been identified that attenuate the effects of HAAs. The most frequently used in vivo method to detect protective effects are adduct measurements; anticarcinogenic dietary factors were identified by aberrant crypt foci assays and liver foci tests with rats. The mechanisms of protection include inactivation of HAAs and their metabolites by direct binding, inhibition of enzymes involved in the metabolic activation of the amines, induction of detoxifying enzymes, and interaction with DNA repair processes. The detection spectrum of conventional in vitro mutagenicity assays with metabolically incompetent indicator cells is limited. These procedures reflect only simple mechanisms such as direct binding of the HAAs to pyrroles and fibers. It has been shown that these compounds are also effective in rodents. More complex mechanisms, namely, interactions with metabolic activation reactions are not adequately represented in in vitro assays with exogenous enzyme homogenates, and false-negative as well as false-positive results may be obtained. More appropriate approaches for the detection of protective effects are recently developed test systems with metabolically competent cells such as the human Hep G2 line or primary hepatocytes. SCGE tests and DNA adduct measurements with laboratory rodents enable the detection of antigenotoxic effects in different organs, including those that are targets for tumor induction by the amines. Medium term assays based on aberrant crypt foci in colon and liver foci tests have been used to prove that certain compounds that prevented DNA damage by HAAs also reduced their carcinogenic effects. These experiments are costly and time consuming and, due to the weak induction capacity of the amines, only pronounced anticar-cinogenic effects can be detected. Over the years, a large bulk of data on HAA protective compounds has accumulated, but only for a few (e.g., fibers, pyrroles, constituents of teas, and lactic acid bacteria) is there sufficient evidence to support the assumption that they are protective in humans as well.