Showing papers in "Archives of Toxicology in 1998"
TL;DR: The extent of absorption of flavonoids is an important unsolved problem in judging their many alleged health effects, and pharmacokinetic studies with dietary quercetin glycosides showed marked differences in absorption rate and bioavailability.
Abstract: Flavonoids are polyphenolic compounds that occur ubiquitously in foods of plant origin. Over 4000 different flavonoids have been described, and they are categorized into flavonols, flavones, catechins, flavanones, anthocyanidins, and isoflavonoids. Flavonoids have a variety of biological effects in numerous mammalian cell systems, as well as in vivo. Recently much attention has been paid to their antioxidant properties and to their inhibitory role in various stages of tumour development in animal studies. Quercetin, the major representative of the flavonol subclass, is a strong antioxidant, and prevents oxidation of low density lipoproteins in vitro. Oxidized low density lipoproteins are atherogenic, and are considered to be a crucial intermediate in the formation of atherosclerotic plaques. This agrees with observations in epidemiological studies that the intake of flavonols and flavones was inversely associated with subsequent coronary heart disease. However, no effects of flavonols on cancer were found in these studies. The extent of absorption of flavonoids is an important unsolved problem in judging their many alleged health effects. Flavonoids present in foods were considered non-absorbable because they are bound to sugars as beta-glycosides. Only free flavonoids without a sugar molecule, the so-called aglycones were thought to be able to pass through the gut wall. Hydrolysis only occurs in the colon by microorganisms, which at the same time degrade flavonoids. We performed a study to quantify absorption of various dietary forms of quercetin. To our surprise, the quercetin glycosides from onions were absorbed far better than the pure aglycone. Subsequent pharmacokinetic studies with dietary quercetin glycosides showed marked differences in absorption rate and bioavailability. Absorbed quercetin was eliminated only slowly from the blood. The metabolism of flavonoids has been studied frequently in various animals, but very few data in humans are available. Two major sites of flavonoid metabolism are the liver and the colonic flora. There is evidence for O-methylation, sulfation and glucuronidation of hydroxyl groups in the liver. Bacterial ring fission of flavonoids occurs in the colon. The subsequent degradation products, phenolic acids, can be absorbed and are found in urine of animals. Quantitative data on metabolism are scarce.
243 citations
TL;DR: HLö 7 may serve as a broad-spectrum reactivator in nerve agent poisoning at doses therapeutically relevant in humans after soman, sarin, cyclosarin, or VX the reactivating potency decreased, and the reactivation of human AChE by oximes was dependent on the organophosphate used.
Abstract: The treatment of poisoning by highly toxic organophosphorus compounds (nerve agents) is unsatisfactory. Until now, the efficacy of new potential antidotes has primarily been evaluated in animals. However, the extrapolation of these results to humans is hampered by species differences. Since oximes are believed to act primarily through reactivation of inhibited acetylcholinesterase (AChE) and erythrocyte AChE is regarded to be a good marker for the synaptic enzyme, the reactivating potency can be investigated with human erythrocyte AChE in vitro. The present study was undertaken to evaluate the ability of various oximes at concentrations therapeutically relevant in humans to reactivate human erythrocyte AChE inhibited by different nerve agents. Isolated human erythrocyte AChE was inhibited with soman, sarin, cyclosarin, tabun or VX for 30 min and reactivated in the absence of inhibitory activity over 5-60 min by obidoxime, pralidoxime, HI 6 or HLo 7 (10 and 30 microM). The AChE activity was determined photometrically. The reactivation of human AChE by oximes was dependent on the organophosphate used. After soman, sarin, cyclosarin, or VX the reactivating potency decreased in the order HLo 7 > HI 6 > obidoxime > pralidoxime. Obidoxime and pralidoxime were weak reactivators of cyclosarin-inhibited AChE. Only obidoxime and HLo 7 reactivated tabun-inhibited AChE partially (20%), while pralidoxime and HI 6 were almost ineffective (5%). Therefore, HLo 7 may serve as a broad-spectrum reactivator in nerve agent poisoning at doses therapeutically relevant in humans.
134 citations
TL;DR: High levels of IMPA appear to correlate with low levels of residual butyrylcholinesterase activity in the samples and vice versa, and in several cases, the doses appeared to be substantially higher than the assumed lethal doses in man.
Abstract: A convenient and rapid micro-anion exchange liquid chromatography (LC) tandem electrospray mass spectrometry (MS) procedure was developed for quantitative analysis in serum of O-isopropyl methylphosphonic acid (IMPA), the hydrolysis product of the nerve agent sarin. The mass spectrometric procedure involves negative or positive ion electrospray ionization and multiple reaction monitoring (MRM) detection. The method could be successfully applied to the analysis of serum samples from victims of the Tokyo subway attack and of an earlier incident at Matsumoto, Japan. IMPA levels ranging from 2 to 135 ng/ml were found. High levels of IMPA appear to correlate with low levels of residual butyrylcholinesterase activity in the samples and vice versa. Based on our analyses, the internal and exposure doses of the victims were estimated. In several cases, the doses appeared to be substantially higher than the assumed lethal doses in man.
113 citations
TL;DR: The data indicate that quercetin reduces cisplatin toxicity in cultured tubular epithelial cells, and the exact mechanism of protection is unclear, though scavenging of free oxygen radicals may play an important role.
Abstract: Quercetin (QC), a polyphenolic compound widely distributed in fruits and vegetables has recently gained interest due to its cisplatin (CP) sensitizing properties in cancer cells. It is currently unknown, whether quercetin also increases the susceptibility of the kidneys to cisplatin toxicity. We studied the effects of various bioflavonoids on CP toxicity in an in vitro model of cultured tubular epithelial cells (LLC-PK1). Viability of LLC-PK1 cells, as assessed by lactate dehydrogenase (LDH) release and MTT-test, was affected by CP (100–400 μM) in a time and dose dependent fashion. Pretreatment of cells with QC for 3 h significantly reduced the extent of cell damage. The protective activity of QC was concentration dependent, starting at 10–25 μM and reaching a plateau between 50 and 100 μM. Other bioflavonoids (catechin, silibinin, rutin) did not diminish cellular injury, even at higher concentrations (100–500 μM). Quercetin itself showed some intrinsic cytotoxicity at concentrations exceeding 75 μM. Our data indicate that quercetin reduces cisplatin toxicity in cultured tubular epithelial cells. The exact mechanism of protection is unclear, though scavenging of free oxygen radicals may play an important role.
106 citations
TL;DR: Results indicate that lipid peroxidation is a very sensitive cellular response to the mycotoxin fumonisin B1 observed at concentrations lower than that required to inhibit cellular synthesis of macromolecules, protein and DNA.
Abstract: The effects of fumonisin B1 (FB1) from Fusarium moniliforme on lipid peroxidation and protein and DNA syntheses were studied in monkey kidney cells (Vero cells). FB1 was found to be a potent inducer of malondialdehyde (MDA), one of the secondary products formed during lipid peroxidation. At 0.14 μM (0.1 μg/ml), FB1 induced 0.496 ± 0.1 nmoles of MDA/mg protein, compared to the control level 0.134 ± 0.01 nmoles of MDA/mg protein (P 14 μM (10 μg/ml) with an IC50 of 33 μM for both protein synthesis and DNA synthesis. These results indicate that lipid peroxidation is a very sensitive cellular response to the mycotoxin fumonisin B1 observed at concentrations lower than that required to inhibit cellular synthesis of macromolecules, protein and DNA. This oxidative damage induced by FB1 concentrations encountered in naturally contaminated foodstuffs and feed might lead to mutagenicity and genotoxicity.
105 citations
TL;DR: Findings indicate that different G proteins, both of which are IAP-sensitive, are present in the rER and Golgi apparatus, and suggest that these G proteins participate in the disturbance of intracellular transport of the secretory ameloblast exposed to fluoride.
Abstract: In enamel fluorosis model rats treated with sodium fluoride, secretory ameloblasts of incisor tooth germs exhibited disruption of intracellular trafficking. We examined whether heterotrimeric G proteins participated in the disruption of vesicular trafficking of the secretory ameloblast exposed to fluoride, using immunoblotting and pertussis toxin (IAP)-induced adenosyl diphosphate (ADP)-ribosylation for membrane fractions of the cell. Immunoblotting of crude membranes, post supernatants of the ameloblast, with anti-Gi3/o and anti-Gs antibodies showed that Gi3 or Go proteins existed in the secretory ameloblast, but Gs protein did not. Immunoblotting of the subcellular membrane fractions indicated that the Gi3 or Go proteins were located in the Golgi membrane, but were not in the rough endoplasmic reticulum (rER) membrane. Autoradiograph of IAP-induced ADP-ribosylation, however, showed the existence of IAP-sensitive G proteins both in rER and Golgi membranes. Fluoride treatment decreased the G proteins bound to both membranes. These findings indicate that different G proteins, both of which are IAP-sensitive, are present in the rER and Golgi apparatus, and suggest that these G proteins participate in the disturbance of intracellular transport of the secretory ameloblast exposed to fluoride.
101 citations
TL;DR: Results suggest that Cu (I) and H2O2 both have important roles in the production of active species in these systems and cause DMPO/·OH formation and help to explain the oxidative damage to DNA observed in the presence of MT and cadmium in vitro.
Abstract: A variety of Cu reconstituted metallothioneins (MTs) containing different amounts of copper ions together with Cd7-MT free of copper were prepared and used in spin trapping experiments designed to show cadmium is not a Fenton active metal. A significant increase of the DMPO/.OH adduct was observed, with increased concentrations of the copper containing MTs, H2O2 enhanced DMPO/.OH adduct formation, catalase and the Cu (I) specific chelating agent bathocuproine, reduced DMPO/.OH adduct formation. These results suggest that Cu (I) and H2O2 both have important roles in the production of active species in these systems and cause DMPO/.OH formation. However, Cd7-MT showed no ability to cause generation of DMPO adducts with H2O2 seeming to indicate cadmium is not a Fenton metal. To test this hypothesis further trapping studies were run with added sulphite and lipid peroxide using both commercial MT and Cd7-MT since cadmium causes peroxidation in vivo. Commercial MT generates radicals with added sulphite and peroxide, Cd7-MT does not, demonstrating that cadmium is not a Fenton metal. These results help to explain the oxidative damage to DNA observed in the presence of MT and cadmium in vitro.
101 citations
TL;DR: Data show that the `non-responsive' guinea-pig expresses active PPARα in the liver at reduced levels, and may be a useful model for exploring the mechanisms underlying the human response to PPs.
Abstract: The peroxisome proliferator class of non-genotoxic rodent hepatocarcinogens cause hepatocyte DNA synthesis, peroxisome proliferation and liver tumours when administered to rats and mice, but fail to induce S-phase or peroxisome proliferation in hepatocytes from other species including guinea-pigs, dogs, and primates including humans. There are compelling data that implicate a nuclear receptor, the peroxisome proliferator-activated receptor-alpha (PPARalpha) as an important mediator of the toxic and carcinogenic effects of peroxisome proliferators (PPs). We were interested to consider the guinea-pig as a possible model for human responses to these compounds. This manuscript describes the isolation of a full-length cDNA encoding PPARalpha from guinea-pig liver that is closely related to receptors identified previously in mouse, rat and human. RNA hybridisation experiments suggested that the livers of the PP-responsive rat and mouse contained relatively high levels of PPARalpha transcripts, whereas in human and guinea-pig liver PPARalpha mRNA was much less abundant. Functional analyses suggested that the guinea-pig PPARalpha was able to be activated by PPs. DNA binding studies using in vitro translated proteins showed that the guinea-pig receptor was able to bind specifically to DNA in the presence of the retinoid X receptor (RXR), and transient transfection assays showed that the guinea-pig PPARalpha was capable of being transcriptionally activated in a concentration-dependent fashion by the PPs Wy-14,643 and nafenopin. Also, in guinea-pig primary hepatocyte cultures, a dominant negative repressor of PPARalpha ablated the suppression of spontaneous apoptosis by PPs. Taken together, these data show that the 'non-responsive' guinea-pig expresses active PPARalpha in the liver at reduced levels, and may be a useful model for exploring the mechanisms underlying the human response to PPs.
96 citations
TL;DR: It is suggested that BPA affects male-specific P450 isoforms in rat liver, and that these changes closely relate to the toxicity of BPA.
Abstract: We examined the effect of bisphenol A (BPA) on microsomal cytochrome P450 (P450) enzymes in rats Rats were treated intraperitoneally with BPA daily for 4 days, at doses of 10, 20, and 40 mg/kg Among the P450-dependent monooxygenase activities, testosterone 2α-hydroxylase (T2AH) and testosterone 6β-hydroxylase (T6BH) activities, which are associated with CYP2C11 and CYP3A2 respectively, were remarkably decreased by 40 mg/kg BPA The levels of the control activities were 13 and 50%, respectively Furthermore, immunoblotting showed that BPA (20 or 40 mg/kg) significantly reduced CYP2C11/6 and CYP3A2/1 protein levels in rat liver microsomes In addition, estradiol 2-hydroxylase (ED2H) and benzphetamine N-demethylase (BZND) activities were significantly decreased by BPA at 20 and 40 mg/kg (by 19–73%) The K
m values for T2AH and T6BH in 20 and 40 mg/kg BPA-treated rats were significantly high compared with that in control rats The V
max for T2AH was dose-dependently decreased by BPA treatment, whereas that of T6BH was only decreased by BPA at 40 mg/kg On the other hand, lauric acid ω-hydroxylase (LAOH) activity was significantly increased by BPA at 20 and 40 mg/kg (15- and 17-fold, respectively) Immunoblot analysis showed that 20 and 40 mg/kg BPA induced CYP4A1/2 protein expression However, the activities 7-ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD), 7-ethoxycoumarin O-deethylase (ECOD), 7-benzyloxyresorufin O-debenzylase (BROD), aminopyrine N-demethylase (APND), chlorzoxazone 6-hydroxylase (CZ6H), erythromycin N-demethylase (EMND), and testosterone 7α-hydroxylase (T7AH) were not affected by BPA at any dose These results suggest that BPA affects male-specific P450 isoforms in rat liver, and that these changes closely relate to the toxicity of BPA
91 citations
TL;DR: Surprisingly, the aliphatic aldehydes and carboxylic acids fit the correlation and with respect to nasal pungency thresholds in man for brief (1–3 s) presentations must be regarded as `nonreactive' compounds.
Abstract: Nasal pungency thresholds (NPT) in man have been determined by Cometto-Muniz and Cain for 44 varied compounds, including esters, aldehydes, ketones, alcohols, carboxylic acids, aromatic hydrocarbons and pyridine. With the exclusion of acetic acid, 43 of these NPT values are well correlated through the general linear free energy equation of Abraham, leading to the algorithm,
where the independent variables are solute descriptors: 2
H is the dipolarity/polarizability, Σ2
H and Σ2
H are the overall or effective hydrogen-bond acidity and basicity, and L
16 is the solute Ostwald solubility coefficient on hexadecane at 25 °C. Surprisingly, the aliphatic aldehydes and carboxylic acids fit the correlation and with respect to nasal pungency thresholds in man for brief (1–3 s) presentations must be regarded as `nonreactive' compounds. It is suggested mere transport of the compound from the air stream to the receptor area largely determines the potency to produce pungency. Various chemical properties of the receptor area are deduced from the coefficients in Eq. i.
86 citations
TL;DR: Results support previous in vivo findings that obidoxime and pralidoxime are insufficient antidotes in cyclosarin poisoning and HLö 7 was shown to be an extremely potent reactivator of human AChE and BChE, which supports its position as a broad-spectrum oxime.
Abstract: Cyclohexylmethylphosphonofluoridate␣(cyclosarin) is a highly toxic organophosphate, which was shown to be rather resistant to conventional oxime therapy. To give more insight into the inhibition, reactivation and aging kinetics, human acetyl-(AChE) and butyrylcholinesterase (BChE) were inhibited by cyclosarin (k2 of 7.4 and 3.8 * 108 M−1 min−1, respectively; pH 7.4, 37 °C) and reactivated with obidoxime, pralidoxime and three experimental oximes. The new oxime HLo 7 (1-[[[4-aminocarbonyl)-pyridinio]-methoxy]-methyl]-2,4-bis-[(hydroxyimino)methyl] pyridinium dimethanesulphonate) was shown to be superior to the other oximes. At oxime concentrations anticipated to be relevant in humans, obidoxime and pralidoxime were extremely weak reactivators of AChE. Aging velocity of BChE was almost fourfold higher compared to AChE (ka of 0.32 h−1 and 0.08 h−1, respectively). A substantial spontaneous reactivation was observed with AChE. These results support previous in vivo findings that obidoxime and pralidoxime are insufficient antidotes in cyclosarin poisoning. By contrast, HLo 7 was shown to be an extremely potent reactivator of human AChE and BChE,␣which supports its position as a broad-spectrum oxime.
TL;DR: A database of 145 volatile organic chemicals for which the sensory irritation potency (RD50) has been reported in mice was used, indicating that the potency of nonreactive chemicals, regardless of their chemical structure, can be estimated using a variety of physicochemical descriptors.
Abstract: We used a database of 145 volatile organic chemicals for which the sensory irritation potency (RD50) has been reported in mice. Chemicals were first separated into two groups: nonreactive and reactive, using Ferguson's rule. This rule suggests that nonreactive chemicals induce their effect via a physical ( p) mechanism (i.e., weak forces or interactions between a chemical and a biological receptor). Therefore, appropriate physicochemical descriptors can be used to estimate their potency. For reactives, a chemical (c) mechanism (i.e., covalent bonding with the receptor) would explain their potency. All chemicals were also separated on the basis of functional groups and subgroups into 24 classifications. Our results indicated that the potency of nonreactive chemicals, regardless of their chemical structure, can be estimated using a variety of physicochemical descriptors. For reactive chemicals, we identified five basic reactivity mechanisms which explained why their potency was higher than that estimated from physicochemical descriptors. We concluded that Ferguson's proposed rule is adequate initially to classify two separate mechanisms of receptor interactions, p vs c. Several physicochemical descriptors can be used to estimate the potency of p chemicals, but chemical reactivity descriptors are needed to estimate the potency for c chemicals. At present, this is the largest database for nonreactive-reactive chemicals in toxicology. Because of the wide variety of c chemicals presented, a semi-quantitative estimate of the potency of new, or not previously evaluated, c chemicals can be arrived at via comparison with those presented and the basic chemical reactivity mechanisms presented.
TL;DR: It is suggested that renal toxicity would not be induced by treatment with minimum amounts of CdCl2 for periods longer than 8 months, although accumulation ofCd might gradually progress.
Abstract: We wished to clarify the relationship between the sensitivity to induce hepato-renal toxicity and the level of cadmium (Cd) in the organs of rats exposed to minimum to large amounts of cadmium chloride (CdCl2). For this purpose, groups of female Sprague-Dawley (SD) rats, each consisting of 24 animals, were fed diet containing CdCl2 at concentrations of 0, 8, 40, 200, and 600 ppm for 2, 4, and 8 months from 5 weeks of age. All surviving rats given 600 ppm Cd were killed at 4␣months because of deterioration of their general condition. Animals of this group showed anemia and decreased hematopoiesis in the bone marrow, in addition to reduction of cancellous bone in their femurs. Hepatotoxicity was observed after 2 months in the groups treated with \(\)200 ppm. By 4 months, the rats in the 600 ppm group had developed periportal liver cell necrosis. Renal toxicity characterized by degeneration of proximal tubular epithelia was apparent in the groups treated with \(\)200 ppm from 2 months, becoming more prominent in the high-dose rats at 4 months. Hepatic accumulation of Cd increased linearly with the duration of treatment. In contrast, the concentration of Cd in the renal cortex of rats treated with 600 ppm reached a plateau level of ∼250 μg/g within the first 2 months. The renal concentration of Cd in the 200 ppm group when renal toxic lesions were first detected at 2 months ranged from 104 to 244 μg/g. No renal lesions were observed in the 40 ppm group after 8 months, despite the presence of 91–183 μg/g of Cd in the kidneys. The results thus suggest that renal toxicity would not be induced by treatment with minimum amounts of CdCl2 for periods longer than 8 months, although accumulation of Cd might gradually progress. A further 2-year feeding study of CdCl2 and Cd-polluted rice is now in progress.
TL;DR: Although there appears to be a species-specific biochemical effect of phosgene exposure for some biochemical indices, measurement of BAL protein in all three species is a better indicator of ensuing edema formation.
Abstract: Phosgene is a highly reactive oxidant gas used in the chemical industry. Phosgene can cause life-threatening pulmonary edema by reacting with peripheral lung compartment tissue components. Clinical evidence of edema is not usually apparent until well after the initial exposure. This study was designed to investigate early signs of acute lung injury in rodents within 45–60 min after the start of exposure. Male mice, rats, or guinea pigs were exposed to 87 mg/m3 (22 ppm) phosgene or filtered room air for 20 min followed by room air washout for 5 min. This concentration-time exposure causes a doubling of lung wet weight within 5 h. After exposure, animals were immediately anesthetized i.p., with pentobarbital. Bronchoalveolar lavage (BAL) was performed and fluid analyzed for total glutathione (GSH), lipid peroxidation thiobarbituric acid reactive substances (TBARS), and protein concentration. Lungs were perfused with saline to remove blood, freeze-snapped in liquid N2, analyzed for tissue GSH, and TBARS. Lung edema was assessed gravimetrically by measuring tissue wet/dry (W/D) weight ratios and tissue wet weights (TWW). W/D and TWW were significantly higher in mice for phosgene vs air (P=0.001, P<0.0001, respectively), but not in rats or guinea pigs. Tissue TBARS was significantly higher in phosgene-exposed guinea pigs, P=0.027; however, BAL TBARS was higher in both rats and guinea pigs, P=0.013 and P=0.006, respectively. Tissue GSH was significantly lower in phosgene-exposed rats and guinea pigs but not mice, whereas BAL GSH was higher in rats, P<0.0001. There were significantly higher BAL protein levels in all phosgene-exposed species: mice, P<0.0001; rats, P<0.0001; and guinea pigs, P=0.002. Although there appears to be a species-specific biochemical effect of phosgene exposure for some biochemical indices, measurement of BAL protein in all three species is a better indicator of ensuing edema formation.
TL;DR: Interestingly, despite the addition of fenfluramine and diethylpropion, two drugs incriminated in the development of valvular heart disease, no cardiac abnormalities were observed.
Abstract: Chinese herbs nephropathy (CHN), a rapidly progressive interstitial fibrosis of the kidney, has been described in approximately 100 young Belgian women who had followed a slimming regimen containing some Chinese herbs. In 4 patients multifocal transitional cell carcinomas (TCC) were observed. Aristolochic acid (AA), suspected as the causal factor of CHN, is a well known carcinogen but its ability to induce fibrosis has never been demonstrated. The objective of this study was to evaluate the latter using doses of AA, durations of intoxication and delays of sacrifice known to yield tumours in rats. We also tested the hypothesis that a possible fibrogenic role of AA was enhanced by the other components of the slimming regimen. Male and female rats were treated orally with 10 mg isolated AA/kg per day for 5 days/week, or with approximately 0.15 mg AA/kg per day 5 days/week contained in the herbal powder together with the other components prescribed in the slimming pills for 3 months. The animals were killed respectively 3 and 11 months later. At sacrifice, animals in both groups had developed the expected tumours but not fibrosis of the renal interstitium, Whether the fibrotic response observed in man is due to species and/or strain related differences in the response to AA or to other factors, remains to be determined. Interestingly, despite the addition of fenfluramine and diethylpropion, two drugs incriminated in the development of valvular heart disease, no cardiac abnormalities were observed.
TL;DR: The data show inducibility of mrp2 gene expression in the liver of primates which may represent an adaptive response aimed at the enhanced biliary elimination of the inducing drugs and/or their metabolites.
Abstract: The multidrug resistance protein 2 (Mrp2) also called canalicular multidrug resistance protein (cMrp) or canalicular multispecific organic anion transporter (cMoat) is a transmembrane export pump located at the canalicular domain of hepatocytes. Mrp2 transports a broad spectrum of organic anions including glucuronides, glutathione conjugates, and organic sulphates into bile. Based on previous observations in rat hepatocytes, the inducibility of mrp2 gene expression in primate liver was investigated in rhesus monkeys treated with tamoxifen or rifampicin. It was found that treatment with tamoxifen (25 mg/kg per day; over 7 days) or rifampicin (15 mg/kg per day; over 7 days) leading to an induction of cytochrome P450 3A4, resulted in a strong increase in mrp2 mRNA in the liver of male and female rhesus monkeys. On the protein level, tamoxifen also was a highly effective inducer, while rifampicin showed some inducing effect in a female and was inactive in a male monkey. In sections of paraffin-embedded liver tissue, immunofluorescence staining confirmed the results of Western blot analysis. Induced Mrp2 was localized to the canalicular domain of the hepatocytes. In conclusion, our data show inducibility of mrp2 gene expression in the liver of primates which may represent an adaptive response aimed at the enhanced biliary elimination of the inducing drugs and/or their metabolites.
TL;DR: Interestingly cytotoxicity as measured by lactate dehydrogenase (LDH) release in the extracellular space was much more pronounced in the VM, hippocampus, and striatum than in the cerebellum, whereas no cytot toxicity was observed in the rest of the brain.
Abstract: Ochratoxin A (OTA) a chlorodihydro-isocoumarin linked through an amide bond to phenylalanine, is a mycotoxin found as a contaminant in foodstuffs and shown to be nephrotoxic, teratogenic, immunosuppressive, genotoxic, mutagenic and carcinogenic in rodents. Ochratoxin A is known to induce teratogenic effects in neonates (rats and mice) exposed in utero, characterised by microcephaly and modification of the brain levels of free amino acids. Since OTA has been found to accumulate in the brain according to the duration of exposure to doses in the range of natural contamination of feedstuffs, experiments were designed to determine more precisely the structural target of OTA in the brain. After intracerebral injection, OTA (403 ng/10 μl) was not found in the following parts of the brain : the frontal cortex (FC), striatum (ST), ventral mesencephalon (VM) and the cerebellum (CB) in contrast to the rest of the brain, probably due to the detection limit of 0.1 ng/g of tissue. However lactate dehydrogenase (LDH) was increased in extracellular space in the VM to a greater extent than in the rest of the brain, indicating that this structure could be one of the targets of OTA in the brain. Contents of free amino acids were morever similarly modified in the VM and in the rest of the brain. Male rats were given OTA (289 μg/kg per 24 h) by gastric intubation for 8 days and the main brain structures analysed for OTA content and cytotoxicity. OTA was found in the following structures in decreasing order: rest of the brain (50.3%), cerebellum (34.4%), VM (5.1%), striatum (3.3%) and hippocampus (2.9%) of the total OTA amount found in the brain, which represents 0.022% to 0.028% of the given dose. Interestingly cytotoxicity as measured by lactate dehydrogenase (LDH) release in the extracellular space was much more pronounced in the VM, hippocampus, and striatum than in the cerebellum, whereas no cytotoxicity was observed in the rest of the brain. Similarly deoxyribonuclease (DNase) activity in relation to possible necrotic cells was increased in the VM and cerebellum. Altogether these results designated the ventral mesencephalon, hippocampus, striatum and cerebellum as the main OTA-targets in the brain of adult rats and excluded the rest of the brain.
TL;DR: The measurement of sarin metabolites in urine is a useful tool for the biological monitoring of exposure to sarin in a patient with sarin poisoning.
Abstract: Sarin metabolites were measured in urine from a patient with sarin poisoning. Two metabolites, methylphosphonic acid (MPA) and isopropylmethylphosphonic acid (iPMPA), were detected by gas chromatography after conversion to volatile derivatives with N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide in the urine from the victim collected on the first day of hospitalization. iPMPA was detected in the urine on the seventh day, but MPA could not be detected in the urine sample. MPA was narrowly detected in the urine collected on the third day. The concentration of iPMPA was estimated on the assumption that the sensitivity of phosphorus was the same as that of MPA. The total excretion of iPMPA and MPA in the urine was 2.1 mg and 0.45 mg, respectively. When all the sarin inhaled was excreted within a week as these two metabolites, the subject was considered to have been exposed to 2.79 mg (0.05 mg/kg) sarin at the incident. Thus, the measurement of sarin metabolites in urine is a useful tool for the biological monitoring of exposure to sarin.
TL;DR: The polymorphic hGSTT1-1 may well play a role in the development of kidney tumours after high and long-term occupational exposure against trichloroethylene and the importance to heed the specific environment at potential target sites in risk assessment is emphasized by these results.
Abstract: Glutathione transferase (GST) GSTT1-1 is involved in the biotransformation of several chemicals widely used in industry, such as butadiene and dichloro methane DCM. The polymorphic hGSTT1-1 may well play a role in the development of kidney tumours after high and long-term occupational exposure against trichloroethylene. Although several studies have investigated the association of this polymorphism with malignant diseases little is known about its enzyme activity in potential extrahepatic target tissues. The known theta-specific substrates methyl chloride (MC) dichloromethane and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) were used to assay GSTT1-1 activity in liver and kidney of rats, mice, hamsters and humans differentiating the three phenotypes (non-conjugators, low conjugators, high conjugators) seen in humans. In addition GSTT1-1 activity towards MC and DCM was determined in human erythrocytes. No GSTT1-1 activity was found in any tissue of non-conjugators (NC). In all organs high conjugators (HC) showed twofold higher activity towards MC and DCM than low conjugators (LC). The activity in human samples towards EPNP was too close to the detection limit to differentiate between the three conjugator phenotypes. GSTT1-1 activity towards MC was two to seven-times higher in liver cytosol than in kidney cytosol. The relation for MC between species was identical in both organs: mouse > HC > rat > LC > hamster > NC. In rats, mice and hamsters GSTT1-1 activity in liver cytosol towards DCM was also two to seven-times higher than in the kidney cytosol. In humans this activity was twice as high in kidney cytosol than in liver cytosol. The relation between species was mouse > rat > HC > LC > hamster > NC for liver, but mouse > HC > LC/rat > hamster/NC for kidney cytosol. The importance to heed the specific environment at potential target sites in risk assessment is emphasized by these results.
TL;DR: Large human trials with natural antioxidants have not provided a uniform support for the hypothesis that antioxidation per se may prevent cancer or coronary heart disease, and interactions between antioxidant systems are only partially understood.
Abstract: It is well documented that diets rich in fruits and vegetables can reduce the risk of most common cancers, and that some food items from this class may be protective against heart disease. Several explanations have been offered, one of which relates to the natural presence of potent antioxidants in plant products. Destructive oxidation of lipids, proteins, DNA, and other important biomolecules, often involving radical chain reactions, affect vital cellular structures and their normal functions. Such processes are involved in the development of cancer as well as heart disease, and it seems logical to assume that antioxidants might be preventive. Large human trials with natural antioxidants have not provided a uniform support, however, for the hypothesis that antioxidation per se may prevent cancer or coronary heart disease (CHD)1. One reason is that other effects, unrelated to antioxidation, may compromise their preventive effects. Another reason may be that many potent antioxidants can also act as pro-oxidants under certain conditions. The interpretation of animal trials is likewise often compromised by the fact that most antioxidants have other physiological effects which might very well explain their protective action or lead to toxic side-effects. In addition, absorption, metabolism and distribution may profoundly influence their antioxidant actions, and not all cellular compartments are equally well protected. Furthermore, interactions between antioxidant systems are only partially understood.
TL;DR: The present study strongly suggested the requirement of persistent increase of 8-OHdG for neoplastic conversion, and a clear sex difference in susceptibility to generation of oxidative stress in kidney DNA was found, in addition to 2-globulin-dependent variation in cell proliferation in the renal tubules.
Abstract: It has been assumed that oxidative damage, including formation of 8-hydroxydeoxyguanosine (8-OHdG) adducts in kidney DNA due to potassium bromate (KBrO3), a renal carcinogen to both sexes of rats, is involved in its mechanisms of tumor induction. However, despite the presumed existence of a repair enzyme(s) for 8-OHdG, there have been no reports demonstrating the changes in adduct levels during medium- or long-term exposure. To elucidate the actual kinetics regarding this parameter during the early stages of KBrO3 carcinogenesis, we measured 8-OHdG levels in kidney DNA together with cell proliferation in renal tubules in both sexes of rats receiving KBrO3 at a dose of 500 ppm in the drinking water for 1, 2, 3, 4, and 13 weeks. Rapid elevation of 8-OHdG levels was noted in treated male rats which persisted until the end of the experiment. Increased cell proliferation in the proximal convoluted tubules was also observed throughout the experimental period, concomitant with alpha2mu-globulin accumulation. Increase in 8-OHdG levels in treated females first became apparent 3 weeks after the start of exposure, with cell proliferation only elevated at the 13-week time point. The present study, employing the same route and dose of KBrO3 known to cause tumors, strongly suggested the requirement of persistent increase of 8-OHdG for neoplastic conversion. Moreover, a clear sex difference in susceptibility to generation of oxidative stress in kidney DNA was found, in addition to alpha2mu-globulin-dependent variation in cell proliferation in the renal tubules.
TL;DR: The data support the hypothesis that the carcinogenicity of nongenotoxic hepatocarcinogens is associated strongly with the ability to perturb hepatocyte growth regulation, suggesting that the growth perturbation may need to exceed a threshold for carcinogenesis.
Abstract: Nongenotoxic rodent hepatocarcinogens do not damage DNA but cause liver tumours in the rat and mouse, associated with the induction of hepatic DNA synthesis. Previously, we have demonstrated that nongenotoxic hepatocarcinogens such as phenobarbitone and the peroxisome proliferator (PP), nafenopin, also suppress rat hepatocyte apoptosis. The nongenotoxic chemicals 1,4-dichlorobenzene (DCB) and the PP, diethylhexyl phthalate (DEHP), both induce high levels of DNA synthesis in rat liver in vivo, but only DEHP is hepatocarcinogenic in this species. Here, we investigate whether the difference in rat carcinogenicity of these two hepatic mitogens may be due to differences in their ability to suppress hepatocyte apoptosis. In rat hepatocytes in vitro, MEHP (the active metabolite of DEHP) induced DNA synthesis 2.5-fold (P = 0.001) and suppressed 10- and 4-fold, respectively both spontaneous (P = 0.0008) and transforming growth factor β1 (TGFβ1)-induced (P = 0.0001) apoptosis. DCB gave a small (1.7-fold) increase in DNA synthesis (P = 0.03) and a small (1.7- to 2-fold) suppression of both spontaneous (P = 0.022) and TGFβ1-induced (P = 0.015) apoptosis. We next analysed the induction of DNA synthesis and the suppression of apoptosis in rat liver in vivo. Both DEHP and DCB were able to induce DNA synthesis although, as seen in vitro, the induction by DCB (4.2-fold; P = 0.023) was less marked than that with DEHP (13.4-fold; P = 0.007). Similarly, DEHP and DCB were both able to suppress rat hepatocyte apoptosis in vivo but the magnitude of the suppression was comparable; apoptosis was reduced to undetectable levels in four out of five animals with DCB and three out of five with DEHP. Since both chemicals suppressed apoptosis and induced DNA synthesis in rat liver but, overall, DCB was less potent, the disparate hepatocarcinogenic potential of these two chemicals could arise from differences in the magnitude of growth perturbation. To test this hypothesis, we repeated the studies in mouse, a species where both DCB and DEHP are hepatocarcinogenic. Both in vitro and in vivo, DCB and DEHP/MEHP were able to suppress apoptosis and induce hepatocyte DNA synthesis in the mouse with comparable potencies. The data support the hypothesis that the carcinogenicity of nongenotoxic hepatocarcinogens is associated strongly with the ability to perturb hepatocyte growth regulation. However, the ability to effect such changes is not unique to nongenotoxic carcinogens and is common to some noncarcinogenic chemicals, such as DCB, suggesting that the growth perturbation may need to exceed a threshold for carcinogenesis.
TL;DR: The results indicate that several different xenobiotic-metabolizing CYPs are present in the human lung, possibly contributing to in situ activation of pulmonary procarcinogens.
Abstract: The purpose of the study was to obtain a comprehensive picture of the expression of cytochrome P450s (CYP) in the human lung, broncho-alveolar macrophages (BAM), and peripheral blood lymphocytes. The methods used were reverse transcriptase-polymerase chain reaction (RT-PCR) with gene-specific primers and immunohistochemistry with specific anti-peptide antibodies. In RT-PCR, CYPs 1A1, 2B6/7, 2E1, 2F1, 3A5 and 4B1 were detected in cDNA prepared from whole lung tissue. BAMs expressed CYPs 1B1, 2B6/7, 2C, 2E1, 2F1, 3A5 and 4B1. These tissues lacked CYPs IA2, 2A6, 2D6 and 3A7. In peripheral blood lymphocytes, only CYP1B1 and CYP2E1 mRNAs were consistently detected. In immunohistochemistry with anti-CYP3A antibodies, epithelial staining of CYP3A5 was observed in 100% of individuals, while only about 20% exhibited CYP3A4 staining. CYP3A5 protein was localized in the bronchial wall, bronchial glands, bronchiolar epithelium, alveolar epithelium, vascular endothelium and alveolar macrophages. The results indicate that several different xenobiotic-metabolizing CYPs are present in the human lung, possibly contributing to in situ activation of pulmonary procarcinogens.
TL;DR: Using this approach, sensory irritation was the only effect induced by CBC at low exposure concentrations, however, bronchoconstriction and pulmonary irritation were superimposed on this effect at higher exposure concentrations.
Abstract: We expanded a previously described rule-based computerized method to recognize the sensory irritating effect of airborne chemicals. Using 2-chlorobenzylchloride (CBC) as a sensory irritant, characteristic modifications of the normal breathing pattern of exposed mice were evaluated by measuring the duration of braking (TB) after inspiration and the resulting decrease in breathing frequency. From the measurement of TB, each breath was then classified as normal (N) or sensory irritation (S). Using increasing exposure concentrations, the classification S increased from ≤2% (equivalent to sham-exposure) to 100% within a narrow exposure concentration range. The potency of CBC was then evaluated by calculating the concentration necessary to produce 50% of the breaths classified as S, i.e., S50. This approach is easier to use than obtaining RD50 (decrease in respiratory frequency by 50%) when high exposure concentrations are difficult to achieve. Detection limits were also established for this bioassay and experiments were conducted to obtain a level of response just around these limits, in order to delineate the practicality of using this bioassay at low exposure concentrations. Using this approach, sensory irritation was the only effect induced by CBC at low exposure concentrations. However, bronchoconstriction and pulmonary irritation were superimposed on this effect at higher exposure concentrations.
TL;DR: It is believed the GST exert a critical role in normal cell house-keeping activities and the extent of altered risk conferred by genotypes is generally 2-3 fold and it is necessary to identify which other genes interact with the GST so that haplotypes associated with 10-20 fold increases in risk can be defined.
Abstract: Allelism has been found in human glutathione S-transferase (GST) genes of the alpha, mu, theta and pi families with the best characterised examples being those in mu class GSTM1 and theta class GSTT1. Isoenzymes encoded by these genes catalyse the detoxification of various reactive toxic and mutagenic compounds including epoxides resulting from the cytochrome P450-mediated metabolism of polycyclic aromatic hydrocarbons as well as lipid and DNA products of oxidative stress (Hayes and Strange, 1995, Smith et al, 1995). Homozygosity for null alleles or those encoding low activity variants are likely therefore, to be associated with a biochemical consequence. However, while accumulating evidence suggests the importance of different GST, it remains unclear precisely which in vivo processes are influenced by these polymorphisms. In this chapter we discuss firstly, a new polymorphism in GSTM3 and secondly, the role of GST polymorphisms in determining cancer susceptibility with particular reference to allelism at GSTMI, GSTT1 and GSTM3 and their interactions with cytochrome P450 (CYP) genotypes in basal cell carcinoma of skin (BCC).
TL;DR: In this paper, enantiomers of α- and β-pinenes were evaluated for their potency in outbred OF1 and NIH/S mice using ASTM E981-84 bioassay.
Abstract: To clarify the existence of a receptor protein for sensory irritants in trigeminal nerve endings, d- [i.e. (+)] and l- [i.e. (−)] enantiomers of α- and β-pinene as models of nonreactive chemicals were evaluated for their potency in outbred OF1 and NIH/S mice using ASTM E981-84 bioassay. All pinenes possess sensory irritation properties and also induced sedation and signs of anaesthesia but had no pulmonary irritation effects. According to the ratio of RD50 (i.e. concentration which causes a 50% decrease in respiratory rate, f ) and vapour pressure (Po), all pinenes are nonreactive chemicals. For nonreactive chemicals, P° and olive oil-gas partition (LOil) can be used to estimate their potency as sensory irritant. Thus, for enantiomers with identical physicochemical properties, the estimated RD50 values are the same. In addition, although α- and β-pinene do not have identical Po and LOil values, their estimated potencies are quite close. However, the experimental results showed that d-enantiomers of pinenes were the most potent as sensory irritants and a difference in potency also exists between α- and β-pinene. RD50 for d-enantiomers of α- and β-pinene were almost equal, 1053 ppm and 1279 ppm in OF1 strain and 1107 ppm and 1419 ppm in NIH/S strain, respectively. Values differed by a factor of ∼4 to 5 from l-β-pinene for which the RD50 was 4663 ppm in OF1 and 5811 ppm in NIH/S mice. RD50 could not be determined for l-α-pinene; this pinene was almost inactive. d-α-pinene seems to best fit the receptor because its experimental RD50 was one-half of the estimated value while for d-β-pinene those values were equal. On the contrary, l-β-pinene was about 3 to 4␣times less potent than estimated. l-α-pinene was only slightly active although it was estimated to be as potent as d-α-pinene. The remarkable difference in potency between l-enantiometers is most likely due to a structural difference between α- and β-pinene: the more flexible β-pinene can bend to fit into the receptor better than the rigid α-pinene. The results showed that the commonly used physicochemical descriptors cannot fully explain the potency of these chemicals; their three-dimensional structure should also be considered. Because of the stereospecificity of pinenes, a target site for nonreactive sensory irritants is most likely a receptor protein containing a chiral lipophilic pocket.
TL;DR: Exposure to Hg vapor might be a suitable procedure to provide an in vivo model with enhanced brain MT, and Gel fractionation revealed that most Hg was in the brain cytosol fraction and thus bound to MT.
Abstract: Metallothionein (MT) is one of the stress proteins which can easily be induced by various kind of heavy metals. However, MT in the brain is difficult to induce because of blood-brain barrier impermeability to␣most heavy metals. In this paper, we have attempted to induce brain MT in rats by exposure to methylmercury (MeHg) or metallic mercury vapor, both of which are known to penetrate the blood-brain barrier and cause neurological damage. Rats treated with MeHg (40 μmol/kg per day × 5 days, p.o.) showed brain Hg levels as high as 18 μg/g with slight neurological signs 10␣days after final administration, but brain MT levels remained unchanged. However, rats exposed to Hg vapor for 7 days showed 7–8 μg Hg/g brain tissue 24 h after cessation of exposure. At that time brain MT levels were about twice the control levels. Although brain Hg levels fell gradually with a half-life of 26 days, MT levels induced by Hg exposure remained unchanged for >2␣weeks. Gel fractionation revealed that most Hg was in the brain cytosol fraction and thus bound to MT. Hybridization analysis showed that, despite a significant increase in MT-I and -II mRNA in brain, MT-III mRNA was less affected. Although significant Hg accumulation and MT induction were observed also in kidney and liver of Hg vapor-exposed rats, these decreased more quickly than in brain. The long-lived MT in brain might at least partly be accounted for by longer half-life of Hg accumulated there. The present results showed that exposure to Hg vapor might be a suitable procedure to provide an in vivo model with enhanced brain MT.
TL;DR: The frequency of this allele supposedly at-risk for lung cancer was significantly higher than in Middle European populations, but lower than in the Far East, and m2 was exclusively found linked with m1 forming allele CYP1A1*2B.
Abstract: The frequency distribution of four cytochrome P4501A1 (CYP1A1) gene mutations was investigated in 271 Turks from southeast Anatolia by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) assay. Allelic linkage of those mutations was proven by peptide nucleic acid-mediated PCR clamping. Mutation m1 (T6235C) forming an MspI restriction site in the 3′-flanking region occurred with 18.1% frequency (95% confidence interval 14.9–21.6%), m2 (A4889G) leading to an Ile/Val exchange in exon 7 had a frequency of 8.9% (6.6–11.6%), and m4 (C4887A; Thr/Asn-exchange also in exon 7) occurred with 5.7% (3.9–8.0%). T5639C (m3) in the 3′-flanking region was not detected. m2 was exclusively found linked with m1 forming allele CYP1A1*2B. The frequency of this allele supposedly at-risk for lung cancer was significantly higher than in Middle European populations, but lower than in the Far East.
TL;DR: The results show that tested 3- and 4-MeSO2 metabolites of PCB congeners reduce thyroid hormone levels much more than PB in rats, and suggests that the metabolites may act as endocrine-disrupters.
Abstract: Male Sprague-Dawley rats received four consecutive intraperitoneal doses of four kinds of methylsulfonyl (MeSO2) metabolites of polychlorinated biphenyl (PCB) congeners: 3-MeSO2-2,2′,3′,4′,5,6-hexachlorobiphenyl (3-MeSO2-CB132); 3-MeSO2-2,2′,3′,4′, 5,5′-hexachlorobiphenyl (3-MeSO2-CB141); 3-MeSO2-2,2′,4′,5,5′,6-hexachlorobiphenyl (3-MeSO2-CB149) and 4-MeSO2-2,2′,4′,5,5′,6-hexachlorobiphenyl (4-MeSO2-CB149). The congeners were major MeSO2-PCBs determined in human milk, liver and adipose tissue, and the aim was to determine their effect on thyroid hormone levels. All four tested MeSO2 metabolites (20 μmol/kg once daily for 4 days) reduced serum total thyroxine levels by 22–44% at a much lower dose than phenobarbital (PB; 431 μmol/kg once daily for 4 days) on days 2, 3, 4 and 7 after the final doses. Total triiodothyronine levels were reduced 37% by treatment with 4-MeSO2-CB149 at day 7. A 30% increase in thyroid weight was produced by 3-MeSO2-CB141 treatment. Total cytochrome P450 content was increased by 3-MeSO2-CB132, 3-MeSO2-CB141 and 3-MeSO2-CB149, but not by 4-MeSO2-CB149. Thus, it is likely that the 3-MeSO2-hexachlorobiphenyls and 4-MeSO2-CB149 could influence the thyroid hormone metabolism by different mechanism(s). The results show that tested 3- and 4-MeSO2 metabolites of PCB congeners reduce thyroid hormone levels much more than PB in rats. Our finding suggests that the metabolites may act as endocrine-disrupters.
TL;DR: The studies suggest the possibility that ACN-induced tumors may be produced by a mode of action involving 8-oxodG, which is not understood, but does not appear to involve lipid peroxidation or disruption of antioxidant defenses.
Abstract: Acrylonitrile (ACN) produces tumors in rats, particularly gliomas of the brain, but tests for genotoxicity have yielded mixed results and no ACN-DNA adducts have been identified in the brain. To examine the possibility that ACN-related brain tumors were not a consequence of binding of ACN to brain DNA, experiments were conducted to investigate possible epigenetic mechanisms. Male Sprague-Dawley rats were exposed to 0, 3, 30, and 300 ppm ACN in drinking water for 21 days, a range that includes doses associated with brain tumorigenesis. In the 30 and 300 ppm ACN groups, 8-oxodeoxyguanosine (8-oxodG) levels were two fold greater than in the controls. Measures of glutathione levels, glutathione peroxidase and catalase were not significantly changed, but cyst(e)ine was somewhat increased. No changes were found in brain cytochrome oxidase activity, which indicates a lack of metabolic hypoxia. Also, no effects on thiobarbituric acid reactive substances were found, indicating a lack of lipid peroxidation. In an additional experiment, male Sprague-Dawley rats were exposed to 0 or 100 ppm ACN in drinking water for 94 days; interim sacrifices were conducted at 3, 10, and 31 days. Levels of brain nuclear DNA 8-oxodG were significantly increased in ACN-exposed rats compared with controls. Another group of animals were given weekly i.v. injections of 5 mg/kg methylnitrosurea and no increases in 8-oxodG were found. These studies suggest the possibility that ACN-induced tumors may be produced by a mode of action involving 8-oxodG. The formation of 8-oxodG is not understood, but does not appear to involve lipid peroxidation or disruption of antioxidant defenses.