Environmental illness: fatigue and cholinesterase activity in patients reporting hypersensitivity to electricity.

The lack of a pathophysiological marker hinders studies on environmental illnesses of unknown origin. Hence, research focused on the identification of such a marker is a priority. This study investigated the nature and a possible etiology of fatigue in hypersensitivity to electricity (the most commonly reported environmental illness in Sweden). The aim was to test the hypothesis that perceived fatigue was due to alterations in cholinesterase activity. The study group consisted of 14 people who reported a hypersensitivity to electricity, including disabling fatigue. We assessed cholinesterase activity three times: twice based on current symptoms reported by the subjects (severe fatigue attributed to electromagnetic fields and absence of this symptom) and once at a randomly selected time. No significant reduction in acetylcholinesterase was identified in any subject. Examined on a group level, no significant reduction in activity was identified at the time of severe fatigue, and no correlation between reported degree of fatigue and cholinesterase activity was observed. Fatigue attributed to electromagnetic fields was nonphysical and showed a significant correlation to difficulties in concentrating. The results do not support the hypothesis that a change in cholinesterase activity mediates fatigue in people reporting hypersensitivity to electricity.

Sex-hormone binding globulin from sheep serum: purification and effects of pregnancy and treatment with exogenous estradiol.

Sex-hormone binding globulin (SHBG) is a protein that binds sex steroids in the serum of many species. SHBG binds androgens and estrogens in humans and primates with high affinity, but behaves as an androgen binding protein in other species. Here we purified SHBG from ewe and ram sera to homogeneity, by a simple and rapid method. The K(D) of the purified protein was found to be 3.63 nM for testosterone and around 600 nM for estradiol. We also studied the effect of pregnancy on SHBG levels in ewes and the effect of exogenous estradiol administration either orally or parenterally on SHBG levels in rams. Basal levels of SHBG in sheep are not affected by pregnancy or exposure to exogenous estradiol. It is concluded that SHBG regulation of expression in ewes and rams differs from that in humans in that it is not affected by estrogen and possibly is species specific.

Hypophysectomy and/or peroxisome proliferators strongly influence the levels of phase II xenobiotic metabolizing enzymes in rat testis.

The objectives of the present work were to determine the influence of hypophysectomy and/or peroxisome proliferators (PP) on certain xenobiotic-metabolizing enzyme activities, i.e. glutathione transferases (GST), glutathione peroxidase (GPX), phenol sulphotransferases (pSULT), phenol UDP-glucuronosyl transferases (pUGT), catalase, NADP(H) quinone oxidoreductase (QR) and epoxide hydrolases (EH) in the rat testes. Adult male rats, hypophysectomized and their sham-operated controls, were treated for 10 days with clofibrate (0.5%), perfluorooctanoic acid (0.05%, PFOA), acetylsalicylic acid (1%, ASA) and di(2-ethylhexyl)phthalate (2%, DEHP) in their diet. The results show that, in addition to both body and testis weight, hypophysectomy caused dramatic changes in most of the xenobiotic-metabolizing enzyme activities, which have been measured here. The most pronounced effects were seen in cytosolic QR (2.2-fold increase), pUGT (95% reduction), pSULT (75% reduction), mitochondrial catalase (75% reduction), microsomal EH (70% reduction) and microsomal GST (55% reduction). Treatment with PP, i.e. perfluorooctanoic acid (PFOA), clofibrate, acetyl salicylic acid (ASA) and di(2-ethylhexyl)phthalate (DEHP) showed varied effects on the xenobiotic-metabolizing enzyme activities, the highest effects (10-60% reduction) were seen in sham-operated animals. These effects were not so pronounced or were not seen in hypophysectomized rats except for the case of PFOA treatment, which caused increases of enzyme activities. The highest increases were seen with microsomal GST (70%), GPX (75%) and cytosolic EH (75%). It is concluded from these experiments that the regulation of several xenobiotic-metabolizing enzymes in the rat testis is affected by the pituitary and/or pituitary hormones and that different peroxisome proliferators have variable effects on the levels of these xenobiotic-metabolizing enzymes. The general trend of reduction in enzyme activities implies that the testis is less protected under conditions that can perturb hormonal status.

Can traditional epidemiology detect cancer risks caused by occupational exposure to pesticides?

In order to investigate the possible relationship between cancer and occupational exposure to pesticides, we reviewed the latest literature of the epidemiological studies in this area coming to the conclusion that, while several studies indicate a link between certain pesticides and certain tumors, this information is still insufficient, and further research on the health consequences of exposure to pesticides is needed. Moreover, provided there is a risk, it is often too limited to be detected by available epidemiological techniques. Therefore, in addition to the epidemiological studies, the development of new biology, gene technology and medical biotechnology methods may significantly enhance the specificity of the epidemiological studies. Thus, the fusion of molecular biology and epidemiology into molecular epidemiology may provide more specific methods for monitoring the occupational dependent carcinogenic risk of individuals and groups.

Chemopreventive activity of very low dose dietary tannic acid administration in hepatoma bearing C3H male mice.

Tannins are plant polyphenols comprising a heterogeneous group of compounds. Tannic acid is a common tannin found in tea, coffee, immature fruits, etc. and it has also been used as a food additive. An increasing body of experimental evidence supports the hypothesis that tannins exert anticarcinogenic activity in chemically induced cancers in animal models. In the present study, tannic acid was administered in very low doses in the drinking water of C3H male mice divided into three groups (75 mg/l, 150 mg/l and 300 mg/l). These animals carry a genetic defect and show a high incidence of spontaneous liver tumors (> 50%) at an age older than 12 months. The results showed a decrease in the overall incidence of hepatic neoplasms (adenomas plus carcinomas): 53.3% of animals in the control group developed hepatic neoplasms versus 33.3% in the group given a low dose of tannic acid, 26.6% in the group given a medium dose and 13.3% in the high dosage group. The difference was more pronounced in the animals with carcinomas: 4.44% of mice who received tannic acid developed carcinomas versus 33.3% of those in the control group. Tannic acid administration did not affect the PCNA labeling index of normal hepatocytes. It is concluded that tannic acid dietary intake in low doses can exert a strong dose-dependent chemoprotective activity against spontaneous hepatic neoplasm development in C3H male mice, most probably through antipromoting mechanisms.

Effects of dietary broccoli on rat testicular xenobiotic metabolizing enzymes.

The effects of vegetables on the activities of various metabolizing enzymes in liver and intestine have been studied intensively, whereas studies on effects on testicular metabolizing enzymes are lacking. The present report is the first describing the effects of dietary broccoli on the activities of a number of xenobiotic metabolizing enzymes from rat testes. Groups of male Wistar rats were fed a semisynthetic diet with 10% (w/w) freeze-dried broccoli for 1 week. Different broccoli samples with varying content of glucosinolates were used. Dietary broccoli significantly increased the activities of two testicular phase II enzymes--glutathione S-transferase (1.6-fold) and UDP-glucuronosyl transferase (1.8-fold). The activities of these enzymes differed significantly depending on the conditions during cultivation of the broccoli, because of differences in the content of glucosinolates and other secondary plant metabolites. The levels of two glutathione S-transferase subunits, rGSTM2 and rGSTA, were determined using Western blotting analysis and the levels of both subunits were reduced in animals fed broccoli grown at low S-fertilizer level. Broccoli did not statistically significantly modulate the activities of the phase I enzymes, epoxide hydrolase or NAD(P)H quinone-oxidoreductase, or the phase II enzyme p-sulphotransferase, or the anti-oxidative enzymes catalase and total glutathione peroxidase in rat testes. In general, dietary broccoli affects phase I and phase II enzyme levels in rat testes much less than found in liver, however, two rat testicular phase II xenobiotic metabolizing enzymes were induced.

DNA adducts in human nasal mucosa and white blood cells from smokers and non-smokers.

The goal of the present study was to measure the levels of DNA adducts in human nasal mucosa cells and in total white blood cells in relation to smoking. DNA was isolated from samples of 20 healthy volunteers (six smokers and 14 non-smokers). The levels of DNA adducts were measured by 32P-postlabelling assay. In smokers the mean DNA adduct levels were 3.3 and 17.0 adducts/10(8) nucleotides in total white blood cells and nasal mucosa cells respectively. The corresponding values in non-smokers were 2.0 and 6.8 adducts/10(8) nucleotides. The mean adduct level was significantly higher in nasal mucosa cells than in total white blood cells both in smokers and non-smokers. The mean adduct levels in smokers' nasal mucosa cells were significantly higher than those in non-smokers. Thus the nasal mucosa cells constituted a sensitive tissue for the determination of cigarette smoking induced DNA adducts. Combining the sensitivity of the 32P-postlabelling assay with the specificity of the nasal mucosa to the airborne chemical exposures, the DNA adduct analysis from human nasal mucosa cells represents a method of choice in the assessment of exposure to airborne carcinogens.

Effects of peroxisome proliferators and/or hypothyroidism on xenobiotic-metabolizing enzymes in rat testis.

The objectives of the present work were to study the effects of certain peroxisome proliferators on xenobiotic-metabolizing enzyme activities in the testes of normal and hypothyroid rats, i.e. phenol sulfotransferases (pST), phenol UDP-glucuronosyl transferases (pUDPGT), glutathione transferases (GST), catalase, epoxide hydrolase (EH), glutathione peroxidase (GPX) and NAD(P)H quinone oxidoreductase (QR). Adult male rats (normal and hypothyroid) were treated for 10 days with clofibrate (0.5%), perfluorooctanoic acid (0.5%, PFOA), acetylsalisylic acid (1%, ASA) and di(2-ethylhexyl)phthalate (2%, DEHP) in their diet. The results show that treatment of normal rats with peroxisome proliferators dramatically affects the activities of xenobiotic-metabolizing enzymes (40-60% reduction). The highest effects are seen in catalase activity (50-60% with PFOA and ASA), pUDPGT (55% with PFOA), pST (55% with PFOA) and QR (50% with DEHP). These effects are not seen or are weaker after induction of hypothyroidism. Taken together, it is concluded that different classes of peroxisome proliferators have different effects on rat testicular xenobiotic-metabolizing enzymes.

DNA adduct formation in the human nasal mucosa as a biomarker of exposure to environmental mutagens and carcinogens.

Human exposure to chemical compounds, often termed xenobiotics, has been linked to a number of enhanced incidences of various neoplasias. A majority of these enter the human body through inhalation. Most xenobiotics are metabolized in the body to more hydrophilic metabolites before excretion in the urine and bile. During this process, promutagens and procarcinogens could be activated and could interact with proteins as well as DNA to form adducts. DNA adducts formed by chemical carcinogens can, therefore, be used as biomarkers of exposure and other host factors. This study that DNA adduct analysis can be performed on cells from human nasal mucosa. Using the nasal lavage procedure performed on 20 healthy volunteers, 5 x 10(5) to 5 x 10(6) cells were obtained from which 5 to 40 micrograms DNA was isolated. DNA adducts were analyzed by the 32-P-postlabeling assay. The DNA adduct levels ranged between 1.4 and 6 adducts/10(8) nucleotides. In addition to its simplicity, the nasal lavage procedure is an inexpensive, noninvasive procedure that requires no anesthetics or special equipment. Moreover, the cells obtained are the first to come in contact with air pollutants. DNA adduct analysis from human nose mucosa cells could therefore be used to develop a technique suitable for the assessment of exposure to chemical carcinogens through inhalation.

Swedish dioxin survey: evaluation of the H-4-II E bioassay for screening environmental samples for dioxin-like enzyme induction.

The H-4-II E enzyme induction bioassay was used for testing both pure reference substances and extracts of wildlife samples. Polychlorinated naphthalenes were found to be as active as enzyme inducers as certain coplanar polychlorinated biphenyls (PCBs). Also a mixture of polybrominated diphenyl ethers (Bromkal 70-5DE) was shown to induce enzyme activity. In extracts of herring, containing polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), bioassay and chemically derived TCDD-equivalents (TEQs) were nearly identical. When extracts containing other types of dioxin-like compounds as well were tested, the bioassay TEQs for most of them agreed well with chemical TEQs calculated for PCDDs, PCDFs and non-ortho PCBs. However, for ringed seal and whitefish, TEQs obtained from the bioassay were higher than those from the chemical analysis. Our results indicate that this bioassay is an excellent complement to chemical residue analysis and a useful tool in understanding the complex interactions of halogenated hydrocarbons. For risk assessment, such results should, however, be used most carefully as they are measured in vitro.

Inhibition of meiotic divisions of rat spermatocytes in vitro by polycyclic aromatic hydrocarbons.

The toxic effects of polycyclic aromatic hydrocarbons (PAH) on spermatogenic cells undergoing meiotic division were investigated in vitro. Toxicity was assayed as alterations in cell nucleus morphology and cell survival and by DNA flow cytometry. Benzo[a]pyrene (BP) and 7,12-dimethylbenz[a]anthracene (DMBA) inhibited the progression of spermatocytes through meiotic division and were highly cytotoxic at concentrations higher than 1 microM. These results were obtained upon addition of a drug-metabolizing system, indicating that the seminiferous tubules lack the enzymes required for the initiation of PAH metabolism. The spindle poisons, e.g., vincristine and Colcemid, a group of direct-acting agents, affected spermatogenesis during meiotic division in a manner similar to that observed with PAH. In contrast, adriamycin did not inhibit meiotic division, although it did induce the formation of meiotic micronuclei as a result of chromosome breakage. It is concluded that low concentrations, i.e., 0.1 microM of PAH, strongly inhibit meiotic division, presumably after metabolic activation to reactive molecules functionally resembling direct-acting alkylating agents. High concentrations of PAH are cytotoxic.

On the identity of the xenobiotic-metabolizing form(s) of cytochrome P-450 in endocrine organs.

The adrenal cortex, the testes and the ovary metabolize polycyclic aromatic hydrocarbons (PAHs), e.g., 7,12-dimethylbenz[a]anthracene (DMBA). These activities have previously been shown to involve the cytochrome P-450 monooxygenase system [18-20]. In attempt to identify the form(s) of cytochrome P-450 involved, microsomes from these endocrine organs were subjected to SDS-gel electrophoresis, followed by immunochemical analysis using the Western blot technique. Antisera raised against the purified rat hepatic cytochrome P-450 isozymes a, b + e, c, d and PB-PCNE were tested. It was concluded that the electrophoretic mobilities of the immunoreactive bands obtained were not identical to the mobilities of the purified isozymes cytochromes P-450 a, b, c, d, e or PB-PCNE. These results indicate that the PAH-metabolizing monooxygenase(s) in these endocrine organs may involve a novel form(s) of cytochrome P-450.

Cell-specific metabolic activation of 7,12-dimethylbenz[a]anthracene in rat testis.

The binding of metabolites of the polycyclic aromatic hydrocarbon (PAH) 7,12-dimethylbenz[a]anthracene (DMBA) to protein in rat testis seminiferous tubules was studied. Treatment of cultured seminiferous tubule segments with DMBA resulted in very little binding to protein, suggesting that the seminiferous epithelium from rat testis lacks the cytochrome P-450-dependent monooxygenase(s) required for DMBA metabolism. In contrast, Leydig cells from rat testis contain monooxygenase systems which catalyze the metabolism of PAH, such as DMBA. This metabolic activation of DMBA was localized in both mitochondria and microsomes derived from Leydig cells and was decreased by inhibitors of the cytochrome P-450 system and by free radical scavengers, suggesting that the metabolism involved both cytochrome P-450 and free radical-dependent pathways. In the presence of whole Leydig cells or microsomes prepared from Leydig cells, the covalent binding of DMBA metabolites to protein of rat testis seminiferous tubules was increased 5- and 13-fold, respectively. These results suggest that DMBA is metabolized primarily in rat testis Leydig cells and that part of the produced metabolites find their way to the seminiferous epithelium, where they undergo further metabolism producing reactive metabolites, possibly cation radicals and diolepoxides, which interfere with the functions of spermatogonia and spermatocytes by modifying key proteins covalently.

Inhibition of stage-specific DNA synthesis in rat spermatogenic cells by polycyclic aromatic hydrocarbons.

Changes in the rate of DNA synthesis in spermatogenic cells after treatment of segments of rat seminiferous tubule at defined stages of epithelial cycle with benzo[a]pyrene (BP) or 7,12-methylbenz[a]anthracene (DMBA) were studied. The incorporation of labeled thymidine into DNA was used as a measure of the rate of DNA synthesis. Very little or no inhibition of DNA synthesis at stages V and VIII of the cycle was observed at BP and DMBA concentrations lower than 100 microM. In contrast, in the presence of added mitochondria and/or microsomes from whole rat testis, 20 microM BP or DMBA inhibited DNA synthesis 5% and 80%, respectively. This inhibition of DNA synthesis was prevented by inhibitors of the cytochrome P-450 system and by free radical scavengers. These results suggest that polycyclic aromatic hydrocarbons (PAH) require metabolic activation in order to inhibit DNA replication in seminiferous tubules. The first step of this biotransformation is cytochrome P-450-dependent and occurs in Leydig cells. However, the metabolites produced in this step may be further metabolized to reactive metabolites by peroxidative pathways in the seminiferous tubules; these latter products may affect DNA replication.

Generation of superoxide anion and lipid peroxidation in different cell types and subcellular fractions from rat testis.

Mitochondria and microsomes from whole rat testis, seminiferous tubules and Leydig cells were investigated with respect to their capacity to generate superoxide anion. In addition, lipid peroxidation by whole testis mitochondria and microsomes was measured. In the presence of NADH and various respiratory inhibitors all three mitochondrial preparations catalyzed the formation of superoxide anion at a rate of 0.27-1.67 nmol/min.mg. This formation was concluded to be confined mainly to the NADH dehydrogenase region of the respiratory chain. Addition of NADPH to whole testis or Leydig cell mitochondria, but not tubule mitochondria, caused an additional formation of superoxide anion, which was unrelated to the respiratory chain, accelerated several-fold by menadione, and presumably catalyzed by NADPH-cytochrome c reductase and cytochrome P-450. Microsomes isolated from whole testis, seminiferous tubules, and Leydig cells generated superoxide anion at rates between 0.19 and 0.44 nmol/min.mg. These rates were also strongly stimulated by menadione. It is likely that both NADPH-cytochrome c reductase and cytochrome P-450 were involved in the microsomal generation of superoxide. Free radical scavengers of various types inhibited both the mitochondrial and microsomal formation of superoxide anion. Lipid peroxidation in whole testis essentially paralleled superoxide anion generation. However, the rate of mitochondrial lipid peroxidation was twice that of the microsomal rate. It is concluded that seminiferous tubules and Leydig cells generate superoxide anion at different rates and by different mechanisms. Together with cytochrome P-450-dependent hydroxylases, e.g., BP and DMBA hydroxylases, this superoxide generation may reflect a potential for cell-specific peroxidative damage in the testis.

Evidence for a free-radical-dependent metabolism of 7,12-dimethylbenz(a)anthracene in rat testis.

Polycyclic aromatic hydrocarbons, e.g., 7,12-dimethylbenz(a)anthracene (DMBA), cause various toxic effects in rat testis. To clarify the mechanism of action of DMBA in adult rat testis microsomes and mitochondria from this organ were investigated in vitro with respect to their capacity to metabolize DMBA. Qualitatively, both preparations showed DMBA-hydroxylase activities which were influenced by cytochrome P-450 inhibitors, chelators, and free-radical scavengers, suggesting that the DMBA metabolism was accounted for by different metabolic pathways in these organelles. Metabolism of DMBA was also accompanied by a pronounced covalent binding to both microsomal and mitochondrial protein, catalyzed primarily by a free-radical mechanism involving free or loosely bound iron which may involve superoxide anion shown to be generated by testis mitochondria. With microsomes covalent binding was markedly enhanced by added horseradish peroxidase but not by hydrogen peroxide whereas the mitochondrial binding was affected neither by added horseradish peroxidase nor by hydrogen peroxide. Antibodies raised against cytochrome P-450 c from rat liver inhibited the microsomal DMBA-hydroxylase but not the mitochondrial DMBA metabolism. It is concluded that the microsomal DMBA conversion and covalent binding are due to a mixture of cytochrome P-450 and free-radical-dependent metabolic pathways whereas the corresponding mitochondrial reaction is due mainly to a free-radical-dependent pathway. However, the data do not allow for a conclusion as to the quantitative importance of these pathways. It is proposed that both pathways may be important in DMBA-dependent testis toxicity but also in polycyclic aromatic hydrocarbon-dependent testis toxicity in general.

Subcellular distribution, catalytic properties and partial purification of epoxide hydrolase in the human adrenal gland.

Epoxide hydrolase in human adrenal gland was characterized with respect to catalytic properties and subcellular distribution. With human adrenal microsomes and the substrates styrene-7,8-oxide, cis-stilbene oxide, estroxide and androstene oxide the specific activities were between 1.9 and 19.0 nmol/min/mg protein. With styrene-7,8-oxide as substrate the apparent Km-value was 0.98 mM and the pH optimum was 9.2. Subcellular fractionation revealed that the bulk of the activity was confined to the endoplasmic reticulum. Different compounds known to influence rodent microsomal epoxide hydrolase activity were also tested on the human adrenal enzyme. 1,1,1-Trichloropropene-2,3-oxide (TCPO) and cyclohexene oxide (CHO) inhibited the activity while benzil and clotrimazole stimulated the activity. Partial purification of human adrenal epoxide hydrolase indicates that its molecular weight is about 51 000 and that its concentration relative total protein in the human adrenal microsomes is about 10%.

Toxic effects of daunorubicin on isolated and cultured heart cells from neonatal rats.

Various aspects of the cardiotoxicity of the anthracycline derivative and antineoplastic drug daunorubicin were investigated using isolated and cultured cells from neonatal rat hearts as a model system. Treatment of the cells with concentrations of daunorubicin of the same order of magnitude as those used in chemotherapy was accompanied by marked toxic effects, e.g. a decreased or abolished contraction, and release of lactate dehydrogenase, pyruvate and oxidized glutathione to the medium. A decreased frequency of contraction appeared to be the most sensitive probe of daunorubicin toxicity, followed by release of pyruvate and oxidized glutathione/lactate dehydrogenase. Daunorubicin and/or its metabolites also bound to cellular protein and DNA. Exposure to daunorubicin was shown to be accompanied by a rapid induction of primarily DT-diaphorase and a slower induction of glutathione transferase. The latter observations are interpreted to indicate a protective role of quinone- and peroxide-metabolizing enzymes, respectively, and support the hypothesis that daunorubicin toxicity involves generation of free radical derivatives, which initiate lipid peroxidation. This conclusion is further substantiated by the demonstration that addition of daunorubicin leads to an increased oxygen consumption.