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Archives of Toxicology (v.82, #4)
Health effects due to endotoxin inhalation (review) by V. Liebers; M. Raulf-Heimsoth; T. Brüning (pp. 203-210).
Endotoxins are ubiquitous in the environment and represent important components of bioaerosols. High exposure occurs in rural environment and at several workplaces (e.g. waste collecting, textile industry etc.). Adverse effects on human health induced by inhalation of endotoxin are described in several studies. Up to now the endotoxin levels are mainly measured using the Limulus amoebocyte-lysate (LAL) assay. This assay is well established, but for a suitable characterization of bioaerosols more parameters are necessary. Additional information, e.g. concerning the pyrogenic activity of organic dust samples may be delivered by whole blood assay. Whereas on the one hand protection measures at workplaces are demanded to avoid lung function impairment due to endotoxin exposure, on the other hand a protective effect of exposure to microbial agents like endotoxins with regard to allergy development has been observed. On the cellular level toll-like receptor 4 (TLR4) and IL-1 receptor as well as surface molecules like CD14 have been shown to play a pivotal role in the endotoxin activation cascade. In this review we summarize the mechanism of endotoxin recognition and its manifold effects on human health.
Keywords: Endotoxin; Health effects; LAL-test; Whole blood assay-allergy
Health effects due to endotoxin inhalation (review) by V. Liebers; M. Raulf-Heimsoth; T. Brüning (pp. 203-210).
Endotoxins are ubiquitous in the environment and represent important components of bioaerosols. High exposure occurs in rural environment and at several workplaces (e.g. waste collecting, textile industry etc.). Adverse effects on human health induced by inhalation of endotoxin are described in several studies. Up to now the endotoxin levels are mainly measured using the Limulus amoebocyte-lysate (LAL) assay. This assay is well established, but for a suitable characterization of bioaerosols more parameters are necessary. Additional information, e.g. concerning the pyrogenic activity of organic dust samples may be delivered by whole blood assay. Whereas on the one hand protection measures at workplaces are demanded to avoid lung function impairment due to endotoxin exposure, on the other hand a protective effect of exposure to microbial agents like endotoxins with regard to allergy development has been observed. On the cellular level toll-like receptor 4 (TLR4) and IL-1 receptor as well as surface molecules like CD14 have been shown to play a pivotal role in the endotoxin activation cascade. In this review we summarize the mechanism of endotoxin recognition and its manifold effects on human health.
Keywords: Endotoxin; Health effects; LAL-test; Whole blood assay-allergy
Alternative methods to safety studies in experimental animals: role in the risk assessment of chemicals under the new European Chemicals Legislation (REACH) by W. Lilienblum; W. Dekant; H. Foth; T. Gebel; J. G. Hengstler; R. Kahl; P.-J. Kramer; H. Schweinfurth; K.-M. Wollin (pp. 211-236).
During the last two decades, substantial efforts have been made towards the development and international acceptance of alternative methods to safety studies using laboratory animals. In the EU, challenging timelines for phasing out of many standard tests using laboratory animals were established in the seventh Amending Directive 2003/15/EC to Cosmetics Directive 76/768/EEC. In continuation of this policy, the new European Chemicals Legislation (REACH) favours alternative methods to conventional in vivo testing, if validated and appropriate. Even alternative methods in the status of prevalidation or validation, but without scientific or regulatory acceptance may be used under certain conditions. Considerable progress in the establishment of alternative methods has been made in some fields, in particular with respect to methods predicting local toxic effects and genotoxicity. In more complex important fields of safety and risk assessment such as systemic single and repeated dose toxicity, toxicokinetics, sensitisation, reproductive toxicity and carcinogenicity, it is expected that the development and validation of in silico methods, testing batteries (in vitro and in silico) and tiered testing systems will have to overcome many scientific and regulatory obstacles which makes it extremely difficult to predict the outcome and the time needed. The main reasons are the complexity and limited knowledge of the biological processes involved on one hand and the long time frame until validation and regulatory acceptance of an alternative method on the other. New approaches in safety testing and evaluation using “Integrated Testing Strategies” (ITS) (including combinations of existing data, the use of chemical categories/grouping, in vitro tests and QSAR) that have not been validated or not gained wide acceptance in the scientific community and by regulatory authorities will need a thorough justification of their appropriateness for a given purpose. This requires the availability of knowledge and experience of experts in toxicology. The challenging deadlines for phasing out of in vivo tests in the Cosmetics Amending Directive 2003/15/EC appear unrealistic. Likewise, we expect that the application of validated alternative methods will only have a small or moderate impact on the reduction of in vivo tests under the regimen of REACH, provided that at least the same level of protection of human health as in the past is envisaged. As a consequence, under safety aspects, it appears wise to consider established in vivo tests to be indispensable as basic tools for hazard and risk assessment with respect to systemic single and repeated dose toxicity, sensitisation, carcinogenicity and reproductive toxicity, especially regarding quantitative aspects of risk assessment such as NOAELs, LOAELs and health-related limit values derived from them. Based on the overall evaluation in this review, the authors are of the opinion that in the short- and mid-term, the strategy of the development of alternative methods should be more directed towards the refinement or reduction of in vivo tests. The lessons learnt during these efforts will provide a substantial contribution towards the replacement initiatives in the long-term.
Keywords: Risk assessment; Chemicals; REACH; Alternative method; In vitro; In vivo; In silico; Validation; Integrated testing strategy
Alternative methods to safety studies in experimental animals: role in the risk assessment of chemicals under the new European Chemicals Legislation (REACH) by W. Lilienblum; W. Dekant; H. Foth; T. Gebel; J. G. Hengstler; R. Kahl; P.-J. Kramer; H. Schweinfurth; K.-M. Wollin (pp. 211-236).
During the last two decades, substantial efforts have been made towards the development and international acceptance of alternative methods to safety studies using laboratory animals. In the EU, challenging timelines for phasing out of many standard tests using laboratory animals were established in the seventh Amending Directive 2003/15/EC to Cosmetics Directive 76/768/EEC. In continuation of this policy, the new European Chemicals Legislation (REACH) favours alternative methods to conventional in vivo testing, if validated and appropriate. Even alternative methods in the status of prevalidation or validation, but without scientific or regulatory acceptance may be used under certain conditions. Considerable progress in the establishment of alternative methods has been made in some fields, in particular with respect to methods predicting local toxic effects and genotoxicity. In more complex important fields of safety and risk assessment such as systemic single and repeated dose toxicity, toxicokinetics, sensitisation, reproductive toxicity and carcinogenicity, it is expected that the development and validation of in silico methods, testing batteries (in vitro and in silico) and tiered testing systems will have to overcome many scientific and regulatory obstacles which makes it extremely difficult to predict the outcome and the time needed. The main reasons are the complexity and limited knowledge of the biological processes involved on one hand and the long time frame until validation and regulatory acceptance of an alternative method on the other. New approaches in safety testing and evaluation using “Integrated Testing Strategies” (ITS) (including combinations of existing data, the use of chemical categories/grouping, in vitro tests and QSAR) that have not been validated or not gained wide acceptance in the scientific community and by regulatory authorities will need a thorough justification of their appropriateness for a given purpose. This requires the availability of knowledge and experience of experts in toxicology. The challenging deadlines for phasing out of in vivo tests in the Cosmetics Amending Directive 2003/15/EC appear unrealistic. Likewise, we expect that the application of validated alternative methods will only have a small or moderate impact on the reduction of in vivo tests under the regimen of REACH, provided that at least the same level of protection of human health as in the past is envisaged. As a consequence, under safety aspects, it appears wise to consider established in vivo tests to be indispensable as basic tools for hazard and risk assessment with respect to systemic single and repeated dose toxicity, sensitisation, carcinogenicity and reproductive toxicity, especially regarding quantitative aspects of risk assessment such as NOAELs, LOAELs and health-related limit values derived from them. Based on the overall evaluation in this review, the authors are of the opinion that in the short- and mid-term, the strategy of the development of alternative methods should be more directed towards the refinement or reduction of in vivo tests. The lessons learnt during these efforts will provide a substantial contribution towards the replacement initiatives in the long-term.
Keywords: Risk assessment; Chemicals; REACH; Alternative method; In vitro; In vivo; In silico; Validation; Integrated testing strategy
Enhanced PON1 activity in the kidneys of cyclophosphamide treated rats may play a protective role as an antioxidant against cyclophosphamide induced oxidative stress by Premila Abraham; Emila Sugumar (pp. 237-238).
Recent studies have shown that paraoxanase (PON1) has protective effect against oxidative stress and hence can act as an antioxidant. A time course study was carried out in order to find out alterations in PON1 activity in cyclophosphamide (CYP) induced renal injury. Eight to ten weeks old female rats were administered CYP at the dose of 150 mg/kg body wt. (i.p.) and sacrificed at 6, 16, or 24 h after treatment. Saline treated rats served as control. CYP exposure for 6 h caused a dramatic increase in PON1 activity (83%), which escalated to 160% at 16 h. The renal PON1 activity reached control values 24 h after treatment with CYP. The renal malondialdehyde level was unaltered 6 h after treatment with CYP and an increase by 35% was observed 16 h after treatment with CYP. The present investigation shows for the first time that an increase in renal PON1 activity is an early biochemical event in cyclophosphamide induced renal damage. It is suggested that this enzyme may have a role within the antioxidant systems of the kidney.
Keywords: Cyclophosphamide; PON1 activity; Renal damage; Rat
Enhanced PON1 activity in the kidneys of cyclophosphamide treated rats may play a protective role as an antioxidant against cyclophosphamide induced oxidative stress by Premila Abraham; Emila Sugumar (pp. 237-238).
Recent studies have shown that paraoxanase (PON1) has protective effect against oxidative stress and hence can act as an antioxidant. A time course study was carried out in order to find out alterations in PON1 activity in cyclophosphamide (CYP) induced renal injury. Eight to ten weeks old female rats were administered CYP at the dose of 150 mg/kg body wt. (i.p.) and sacrificed at 6, 16, or 24 h after treatment. Saline treated rats served as control. CYP exposure for 6 h caused a dramatic increase in PON1 activity (83%), which escalated to 160% at 16 h. The renal PON1 activity reached control values 24 h after treatment with CYP. The renal malondialdehyde level was unaltered 6 h after treatment with CYP and an increase by 35% was observed 16 h after treatment with CYP. The present investigation shows for the first time that an increase in renal PON1 activity is an early biochemical event in cyclophosphamide induced renal damage. It is suggested that this enzyme may have a role within the antioxidant systems of the kidney.
Keywords: Cyclophosphamide; PON1 activity; Renal damage; Rat
Perfluorooctanoic acid-induced hepatic toxicity following 21-day oral exposure in mice by Hee-Young Son; Sang-Hyun Kim; Hong-In Shin; Han Ik Bae; Jae-Ho Yang (pp. 239-246).
Perfluorooctanoic acid (PFOA) is a member of the perfluoroalkyl acids that have wide commercial applications and is a widespread pollutant of toxicological importance that has been detected in environmental matrices. The NOAEL and LOAEL of PFOA in rodent were reported 1 and 10 ppm, respectively. The current study characterizes the hepatic toxicities of PFOA in mice. Male ICR mice were exposed continuously to 0, 2, 10, 50 and 250 ppm of PFOA in drinking water for 21 days. Food and water consumption decreased in mice exposed to 250 ppm of PFOA. Mean body weight gain was reduced in mice exposed to 50 and 250 ppm of PFOA. The size and relative weight of the liver increased dose-dependently in PFOA-treated mice. Serum enzyme activities, alanine aminotransferase and aspartate aminotransferase, increased in mice exposed to PFOA in a dose-dependent manner. In the histopathological evaluation, the liver of PFOA-treated mice showed remarkable hepatocytomegaly and acidophilic cytoplasm. At the high doses of PFOA, diffuse hepatic damage by multifocal coagualation and liquefaction necrosis were noted. In contrast to the remarkable change of liver, the kidney had little change. The size and relative weights of the kidney, biomarkers of kidney damage (blood urea nitrogen, creatinine), and histopathological changes had no differences between PFOA-untreated and PFOA-treated mice. In conclusion, our results demonstrate that PFOA causes a toxic effect on the liver but not to the kidney.
Keywords: Perfluorooctanoic acid; Drinking water; Liver; Kidney; Hepatotoxicity
Perfluorooctanoic acid-induced hepatic toxicity following 21-day oral exposure in mice by Hee-Young Son; Sang-Hyun Kim; Hong-In Shin; Han Ik Bae; Jae-Ho Yang (pp. 239-246).
Perfluorooctanoic acid (PFOA) is a member of the perfluoroalkyl acids that have wide commercial applications and is a widespread pollutant of toxicological importance that has been detected in environmental matrices. The NOAEL and LOAEL of PFOA in rodent were reported 1 and 10 ppm, respectively. The current study characterizes the hepatic toxicities of PFOA in mice. Male ICR mice were exposed continuously to 0, 2, 10, 50 and 250 ppm of PFOA in drinking water for 21 days. Food and water consumption decreased in mice exposed to 250 ppm of PFOA. Mean body weight gain was reduced in mice exposed to 50 and 250 ppm of PFOA. The size and relative weight of the liver increased dose-dependently in PFOA-treated mice. Serum enzyme activities, alanine aminotransferase and aspartate aminotransferase, increased in mice exposed to PFOA in a dose-dependent manner. In the histopathological evaluation, the liver of PFOA-treated mice showed remarkable hepatocytomegaly and acidophilic cytoplasm. At the high doses of PFOA, diffuse hepatic damage by multifocal coagualation and liquefaction necrosis were noted. In contrast to the remarkable change of liver, the kidney had little change. The size and relative weights of the kidney, biomarkers of kidney damage (blood urea nitrogen, creatinine), and histopathological changes had no differences between PFOA-untreated and PFOA-treated mice. In conclusion, our results demonstrate that PFOA causes a toxic effect on the liver but not to the kidney.
Keywords: Perfluorooctanoic acid; Drinking water; Liver; Kidney; Hepatotoxicity
Cytotoxicity and apoptosis induced by fumonisin B1, beauvericin and ochratoxin A in porcine kidney PK15 cells: effects of individual and combined treatment by Maja Šegvić Klarić; Lada Rumora; Danica Ljubanović; Stjepan Pepeljnjak (pp. 247-255).
The objective of this study was to determine individual and combined effects of fumonisin B1 (FB1), beauvericin (BEA) and ochratoxin A (OTA) on porcine kidney epithelial PK15 cell survival by measuring lactate dehydrogenase (LDH) activity, apoptotic index and caspase-3 activity. Cells were treated with 0.05, 0.5 and 5 μg/ml of each mycotoxin or with the combinations of two or all three mycotoxins for 24 and 48 h. Changes in LDH and caspase-3 activity, and in apoptotic index showed that the cytotoxic and apoptotic effects of these mycotoxins were concentration- and time- dependent. Significant increase of LDH activity was observed after 48 h of exposure to the highest concentration of FB1 (45%), BEA (84%) and OTA (77%), as compared to control. OTA increased caspase-3 activity after 24 h of treatment with 0.5 μg/mL (84%), while BEA (319%) and FB1 (419%) significantly affected this enzyme activity after 48 h (P < 0.05). Increase of caspase-3 activity preceded significant morphological apoptotic changes, which were detected after 48 h of exposure to a single toxin. Combined treatment with FB1, BEA and OTA resulted mostly in additive effects on LDH activity, and additive and synergistic effects on caspase-3 activity and apoptotic index.
Keywords: Fumonisin B1 ; Beauvericin; Ochratoxin A; Necrosis; Apoptosis; Caspase-3; Mycotoxin interactions
Cytotoxicity and apoptosis induced by fumonisin B1, beauvericin and ochratoxin A in porcine kidney PK15 cells: effects of individual and combined treatment by Maja Šegvić Klarić; Lada Rumora; Danica Ljubanović; Stjepan Pepeljnjak (pp. 247-255).
The objective of this study was to determine individual and combined effects of fumonisin B1 (FB1), beauvericin (BEA) and ochratoxin A (OTA) on porcine kidney epithelial PK15 cell survival by measuring lactate dehydrogenase (LDH) activity, apoptotic index and caspase-3 activity. Cells were treated with 0.05, 0.5 and 5 μg/ml of each mycotoxin or with the combinations of two or all three mycotoxins for 24 and 48 h. Changes in LDH and caspase-3 activity, and in apoptotic index showed that the cytotoxic and apoptotic effects of these mycotoxins were concentration- and time- dependent. Significant increase of LDH activity was observed after 48 h of exposure to the highest concentration of FB1 (45%), BEA (84%) and OTA (77%), as compared to control. OTA increased caspase-3 activity after 24 h of treatment with 0.5 μg/mL (84%), while BEA (319%) and FB1 (419%) significantly affected this enzyme activity after 48 h (P < 0.05). Increase of caspase-3 activity preceded significant morphological apoptotic changes, which were detected after 48 h of exposure to a single toxin. Combined treatment with FB1, BEA and OTA resulted mostly in additive effects on LDH activity, and additive and synergistic effects on caspase-3 activity and apoptotic index.
Keywords: Fumonisin B1 ; Beauvericin; Ochratoxin A; Necrosis; Apoptosis; Caspase-3; Mycotoxin interactions
Induction of micronuclei in V79 cells by the anabolic doping steroids tetrahydrogestrinone and trenbolone by Susanne B. Dorn; Hermann M. Bolt; Mario Thevis; Patrick Diel; Gisela H. Degen (pp. 257-263).
The synthetic steroid tetrahydrogestrinone is a new “designer drug” and was recently detected to be illegally used in sports. It is chemically closely related to trenbolone that is known as an animal growth promoter. The potencies of trenbolone, tetrahydrogestrinone and testosterone to induce micronuclei in V79 cells in vitro were determined. CREST analysis was employed to differentiate between aneugenic or clastogenic mechanisms. Cytotoxicity and an influence on the cell cycle were assessed in parallel. Incubations with testosterone, at concentrations between 3 and 300 μM, failed to induce micronuclei. By contrast, tetrahydrogestrinone and trenbolone increased the rate of micronuclei significantly, up to a doubling of the micronuclei rate of untreated controls. Tetrahydrogestrinone and trenbolone displayed a bell-shaped dose-response curve, with maximal effects observed at 3 and 30 μM, respectively. The micronuclei induced by tetrahydrogestrinone and trenbolone were predominantly kinetochor (CREST) positive, pointing to an aneugenic mode of action. This may be related to the specific structure of both molecules with a system of activated double bonds. As the genotoxic effect of tetrahydrogestrinone at a chromosomal level appears at a low concentration range, it cannot be ruled out that tetrahydrogestrinone presents a genotoxic hazard on a chromosomal level under conditions of its current misuse in sports.
Keywords: Trenbolone; Tetrahydrogestrinone; Testosterone; Anabolic steroids; Doping; Sport; Genotoxicity; Micronucleus assay
Induction of micronuclei in V79 cells by the anabolic doping steroids tetrahydrogestrinone and trenbolone by Susanne B. Dorn; Hermann M. Bolt; Mario Thevis; Patrick Diel; Gisela H. Degen (pp. 257-263).
The synthetic steroid tetrahydrogestrinone is a new “designer drug” and was recently detected to be illegally used in sports. It is chemically closely related to trenbolone that is known as an animal growth promoter. The potencies of trenbolone, tetrahydrogestrinone and testosterone to induce micronuclei in V79 cells in vitro were determined. CREST analysis was employed to differentiate between aneugenic or clastogenic mechanisms. Cytotoxicity and an influence on the cell cycle were assessed in parallel. Incubations with testosterone, at concentrations between 3 and 300 μM, failed to induce micronuclei. By contrast, tetrahydrogestrinone and trenbolone increased the rate of micronuclei significantly, up to a doubling of the micronuclei rate of untreated controls. Tetrahydrogestrinone and trenbolone displayed a bell-shaped dose-response curve, with maximal effects observed at 3 and 30 μM, respectively. The micronuclei induced by tetrahydrogestrinone and trenbolone were predominantly kinetochor (CREST) positive, pointing to an aneugenic mode of action. This may be related to the specific structure of both molecules with a system of activated double bonds. As the genotoxic effect of tetrahydrogestrinone at a chromosomal level appears at a low concentration range, it cannot be ruled out that tetrahydrogestrinone presents a genotoxic hazard on a chromosomal level under conditions of its current misuse in sports.
Keywords: Trenbolone; Tetrahydrogestrinone; Testosterone; Anabolic steroids; Doping; Sport; Genotoxicity; Micronucleus assay
Reconstruction of N-acetyltransferase 2 haplotypes using PHASE by Klaus Golka; Meinolf Blaszkewicz; Mirabutaleb Samimi; Hermann M. Bolt; Silvia Selinski (pp. 265-270).
The genotyping of N-acetyltransferase 2 (NAT2) by PCR/RFLP methods yields in a considerable percentage ambiguous results. To resolve this methodical problem a statistical approach was applied. PHASE v2.1.1, a statistical program for haplotype reconstruction was used to estimate haplotype pairs from NAT2 genotyping data, obtained by the analysis of seven single nucleotide polymorphisms relevant for Caucasians. In 1,011 out of 2,921 (35%) subjects the haplotype pairs were clearcut by the PCR/RFLP data only. For the majority of the data the applied method resulted in a multiplicity (2–4) of possible haplotype pairs. Haplotype reconstruction using PHASE v2.1.1 cleared this ambiguity in all cases but one, where an alternative haplotype pair was considered with a probability of 0.029. The estimation of the NAT2 haplotype is important because the assignment of the NAT2 alleles *12A, *12B, *12C or *13 to the rapid or slow NAT2 genotype has been discussed controversially. A clear assignment is indispensable in surveys of human bladder cancer caused by aromatic amine exposures. In conclusion, PHASE v2.1.1 software allowed an unambiguous haplotype reconstruction in 2,920 of 2,921 cases (>99.9%).
Keywords: PHASE v2.1.1; NAT2 genotyping; Single nucleotide polymorphism; Haplotype reconstruction
Reconstruction of N-acetyltransferase 2 haplotypes using PHASE by Klaus Golka; Meinolf Blaszkewicz; Mirabutaleb Samimi; Hermann M. Bolt; Silvia Selinski (pp. 265-270).
The genotyping of N-acetyltransferase 2 (NAT2) by PCR/RFLP methods yields in a considerable percentage ambiguous results. To resolve this methodical problem a statistical approach was applied. PHASE v2.1.1, a statistical program for haplotype reconstruction was used to estimate haplotype pairs from NAT2 genotyping data, obtained by the analysis of seven single nucleotide polymorphisms relevant for Caucasians. In 1,011 out of 2,921 (35%) subjects the haplotype pairs were clearcut by the PCR/RFLP data only. For the majority of the data the applied method resulted in a multiplicity (2–4) of possible haplotype pairs. Haplotype reconstruction using PHASE v2.1.1 cleared this ambiguity in all cases but one, where an alternative haplotype pair was considered with a probability of 0.029. The estimation of the NAT2 haplotype is important because the assignment of the NAT2 alleles *12A, *12B, *12C or *13 to the rapid or slow NAT2 genotype has been discussed controversially. A clear assignment is indispensable in surveys of human bladder cancer caused by aromatic amine exposures. In conclusion, PHASE v2.1.1 software allowed an unambiguous haplotype reconstruction in 2,920 of 2,921 cases (>99.9%).
Keywords: PHASE v2.1.1; NAT2 genotyping; Single nucleotide polymorphism; Haplotype reconstruction