Jan Krijt

Biochemical Markers for Assessing Aquatic Contamination

Biochemical markers, specifically enzymes of the first phase of xenobiotic transformation - cytochrome P450 and ethoxyresorufin-O-deethylase (EROD) - were used to determine the quantities of persistent organic pollutants (POPs) in fish muscle (PCB, HCB, HCH, OCS, DDT). Eight rivers were monitored (Orlice, Chrudimka, Cidlina, Jizera, Vltava, Ohře and Bílina; and the River Blanice was used as a control). The indicator species selected was the chub (Leuciscus cephalus L.). There were no significant differences in cytochrome P450 content between the locations monitored. The highest concentration of cytochrome P450 in fish liver was in the Vltava (0.241 nmol mg-1 protein), and the lowest was in the Orlice (0.120 nmol mg-1 protein). Analysis of EROD activity showed a significant difference between the Blanice and the Vltava (P< 0.05), and also between the Orlice and the Vltava (P< 0.01), the Orlice and the Bílina (P< 0.01), and the Orlice and the Ohře (P< 0.05). The highest EROD activity in fish liver was in the Vltava (576.4 pmol min-1 mg-1 protein), and the lowest was in the Orlice (63.05 pmol min-1 mg-1 protein). In individual locations, results of chemical monitoring and values of biochemical markers were compared. A significant correlation (P< 0.05) was found between biochemical markers and OCS, and PCB. Among the tributaries studied those that contaminated the Elbe most were the Vltava and the Bílina. These tributaries should not be considered the main sources of industrial contamination of the River Elbe, because the most important contamination sources were along the river Elbe itself.

Organic Pollutant Contamination of the River Tichá Orlice as Assessed by Biochemical Markers

·iroka Z. , J . Kri j t , T. Randak, Z. Svobodova, G. Pe‰kova, J . Fuksa, J . Haj‰ lova, J . Jarkovsk , M. Janska: Organic Pollutant Contamination of the River Elbe as Assessed by Biochemical Markers. Acta Vet Brno 2005, 74: 293-303. The aim of the study was to assess contamination of the River Elbe basin using selected biochemical markers. Biochemical markers selected were enzymes of the first stage of xenobiotic transformation, namely cytochrome P450 (CYP 450) and ethoxyresorufin-O-deethylase (EROD). The results were correlated with the most important inducers of the enzymes, i.e. polychlorinated biphenyls (PCB) concentrations in muscle tissue of fish, polyaromatic hydrocarbons (PAH) values in bottom sediments and 1-hydroxypyrene (1-OHPY) values in fish bile (terminal metabolite of PAH, or, rather, of one of them, i.e. pyrene), which were determined during the chemical monitoring of the River Elbe basin. The indicator species selected was chub (Leuciscus cephalus L.), which was captured at ten locations in the River Elbe basin. A comparison between the EROD activity and the CYP 450 content along the longitudinal profile of the Elbe showed a significant correlation at the level of significance of p < 0.05. The highest EROD activity levels in the liver were ascertained in Zelain (341 pmol·min-1·mg-1), Valy (263.2 pmol·min-1·mg-1) and Lysa nad Labem (179.17 pmol·min-1·mg-1). In Blanice (control location), EROD activity was significantly lower than in any of the other locations studied (p < 0.05). The study failed to produce an unambiguous proof of any correlations between detoxification enzyme activity (CYP 450 and EROD) in the liver and their two important inducers (PCB and PAHs). The possibility that other substances causing activation or inhibition of detoxification enzymes were in play is also discussed. Cytochrome P450, EROD, Leuciscus cephalus L., liver, PCB, PAH, 1-hydroxypyrene The River Elbe is one of the most important European rivers (total length 1103.5 km). Its extensive basin of a total of 148 268 km2 lies on the territory of two countries, i.e. the Czech Republic (51 336 km2) and Germany (96 932 km2). Intensive research of the Czech and German reaches of the Elbe started in 1991 under the Elbe I (1991 1994) project, and continued with the Elbe II (1995 1998) and Elbe III (1999 2002) projects. In those projects, large quantities of data regarding sources of pollution, chemical monitoring of hazardous substances in various components of the aquatic environment, water quality, etc. were collected and evaluated (Nesmurak 1994; BlaIkova et al. 1998; BlaIkova 2002). To enhance the relevance of results obtained by chemical monitoring, it is, however, also ACTA VET. BRNO 2005, 74: 293–303 Address for correspondence: ·iroka Zuzana Department of Public Veterinary Health and Toxicology University of Veterinary and Pharmaceutical Sciences Brno Palackeho 1-3, 612 42 Brno, Czech Republic Phone: +420 541 562 780 E-mail: sirokaz@vfu.cz http://www.vfu.cz/acta-vet/actavet.htm necessary to assess the effects of anthropogenic pollution of the aquatic environment on fish. One of possible ways of assessing such effects is the use of biochemical markers of contamination. They are measurable biochemical parameters responding usually to substances with the same mechanism of toxic effect. That means they are not, with some exceptions, specific for individual xenobiotic substances. Their advantage lies in their ability to provide comprehensive information on the effects of pollution, i.e. to reflect synergic or antagonistic effects of individual components contributing to pollution. In 2003, the Elbe IV Project was started. For reasons mentioned above, chemical monitoring in fish was complemented with assay of biochemical contamination markers. Attention focused primarily on the enzymes of the first phase of xenobiotics conversion, i.e. cytochrome P450 and ethoxyresorufin-O-deethylase (EROD). Cytochrome P450 is an important biochemical marker of surface water contamination with some industrial and agricultural pollutants (Stegeman and Lech 1991). It is now believed that the most useful is the 1A family of cytochrome P450 (Machala et al. 1997; Schlenk and Di Giul io 2002). The most potent inducers for that isoform are substances from the groups of polychlorinated biphenyls (PCB), polycyclic aromatic hydrocarbons (PAH), nitrated polycyclic aromatic hydrocarbons (NPAH) and dioxins (e.g. 2, 3, 7, 8 TCDD) (White et al. 1997; Nilsen et al. 1998; Jung et al. 2001; Schlenk and Di Giul io 2002). On the other hand, chronic exposure to these contaminants can cause a lack of CYP1A induction response (Brammell et al. 2004), and also assessment of CYP1A at the time of spawning can influence its level, because estrogens can decrease CYP1A induction (Elskus 2004). Male fish seem to be more sensitive to PAH and PCB than female fish (McArdle et al. 2004). The induction of the CYP1A family is mediated by the aryl hydrocarbon receptor (AhR) (Bil l iard 2002). Following its interaction with xenobiotic substances, it is carried to the nucleus where it is the cause of enhanced expression of genes for CYP1A and, subsequently, of increased synthesis of cytochrome proteins. The potential toxicity of pollutants depends on their affinity to the AhR. The CYP1As are also responsible for the metabolic activation of most of the known promutagens and procarcinogens, and its elevated levels are responsible for such negative effects as cocarcinogenesis, immunotoxicity and reproduction disorders (Lewis et al. 2003; Carlson et al. 2004). The model CYP1A activity is the enzyme ethoxyresorufin-O-deethylase (EROD), with its ability to convert substrates to products demonstrating fluorescence, which can then be measured. This enzyme is an important biochemical marker of contamination. The aim of the study presented here was to use the assessment of biochemical markers cytochrome P450 and EROD in the livers of the indicator fish species (Leuciscus cephalus L.) to evaluate contamination levels in various locations within the River Elbe basin. Results of chemical monitoring relevant for the above contamination markers are also outlined and correlated in the paper. They were PCB concentrations in chub muscle tissues, concentrations of 1-hydroxypyrene (1-OHPY) in chub bile samples (i.e. the final metabolite of PAHs, or rather of pyrene), and PAH concentrations in bottom sediments in the locations studied. Materials and Methods Animals and Sampling The chub (Leuciscus cephalus L.) was selected as the most suitable indicator species. The chub is a common freshwater cyprinid species that inhabits both clean and polluted rivers (Baru‰ et al. 1995). The fish were captured with the use of a diesel-electric generator in 10 locations in the River Elbe basin. The locations studied were Podoli and Zelain at the River Vltava, a tributary to the Elbe, and Verdek, Numaice, Valy, Lysa nad Labem, Obfiistvi, Duain and Hfiensko along the River Elbe. The control location was upstream of the Husinec water reservoir at the river Blanice in the Vltava basin (Fig. 1). The fish were captured in July 2003 (average water temperature 21.4 °C). In the control location, fish were captured in September 2003 (water temperature 15 °C). In each location, eight chub (both males and females) were captured (except in Lysa nad Labem where only 3 chub were captured). The chub 294

Liver hemojuvelin protein levels in mice deficient in matriptase-2 (Tmprss6).

Mutations of the TMPRSS6 gene, encoding the serine protease matriptase-2, lead to iron-refractory iron deficiency anemia. Matriptase-2 is a potent negative regulator of hepcidin. Based on in vitro data, it has recently been proposed that matriptase-2 decreases hepcidin synthesis by cleaving membrane hemojuvelin, a key protein of the hepcidin-regulatory pathway. However, in vivo evidence for this mechanism of action of matriptase-2 is lacking. To investigate the hemojuvelin-matriptase-2 interaction in vivo, an immunoblot assay for liver membrane hemojuvelin was optimized using hemojuvelin-mutant mice as a negative control. In wild-type mice, two hemojuvelin-specific bands of 35kDa and 20kDa were detected in mouse liver membrane fraction under reducing conditions; under non-reducing conditions, a single band of approximately 50kDa was seen. Phosphatidylinositol-specific phospholipase C treatment confirmed binding of the detected protein to the cell membrane by a glycosylphosphatidylinositol anchor, indicating that the major form of mouse liver membrane hemojuvelin is a glycosylphosphatidylinositol-bound heterodimer. Unexpectedly, comparison of liver homogenates from Tmprss6+/+ and Tmprss6-/- mice revealed significantly decreased, rather than increased, hemojuvelin heterodimer content in Tmprss6-/- mice. These data do not provide direct support for the concept that matriptase-2 cleaves membrane hemojuvelin and may indicate that, in vivo, the role of matriptase-2 in the regulation of hepcidin gene expression is more complex.

Effect of diphenyl ether herbicides and oxadiazon on porphyrin biosynthesis in mouse liver, rat primary hepatocyte culture and HepG2 cells.

The effects of the herbicides fomesafen, oxyfluorfen, oxadiazon and fluazifop-butyl on porphyrin accumulation in mouse liver, rat primary hepatocyte culture and HepG2 cells were investigated. Ten days of herbicide feeding (0.25% in the diet) increased the liver porphyrins in male C57B1/6J mice from 1.4±0.6 to 4.8±2.1 (fomesafen) 16.9±2.9 (oxyfluorfen) and 25.9±3.1 (oxadiazon) nmol/g wet weight, respectively. Fluazifop-butyl had no effect on liver porphyrin metabolism. Fomesafen, oxyfluorfen and oxadiazon increased the cellular porphyrin content of rat hepatocytes after 24 h of incubation (control, 3.2 pmol/mg protein, fomesafen, oxyfluorfen and oxadiazon at 0.125 mM concentration 51.5, 54.3 and 44.0 pmol/mg protein, respectively). The porphyrin content of HepG2 cells increased from 1.6 to 18.2, 10.6 and 9.2 pmol/mg protein after 24 h incubation with the three herbicides. Fluazifop-butyl increased hepatic cytochrome P450 levels and ethoxy- and pentoxyresorufin O-dealkylase (EROD and PROD) activity, oxyfluorfen increased PROD activity. Peroxisomal palmitoyl CoA oxidation increased after fomesafen and fluazifop treatment to about 500% of control values both in mouse liver and rat hepatocytes. Both rat hepatocytes and HepG2 cells can be used as a test system for the porphyrogenic potential of photobleaching herbicides.

Identification of very-long-chain fatty acids in rat and mouse Harderian gland lipids by capillary gas chromatography—mass spectrometry

Lipids of Harderian ophthalmic gland were separated by means of thin-layer chromatography with flame ionization detection in an latroscan apparatus. Wax ester and polar lipids (phosphatidylethanolamine and phosphatidylcholine) were detected as the main lipids in rats and glyceryl ether diester and both polar lipids were the main lipids in mice. Fatty acids were determined in individual lipid classes by means of gas chromatography and gas chromatography-mass spectrometry on capillary columns. The content of fatty acids, the positional isomers of monoenoic acids being predominantly C18, C20 and C22, is most interesting. Very-long-chain fatty acids, saturated fatty acids up to C30 and even monoenoic acids up to C28 were detected. Branched-chain fatty acids, predominantly iso and anteiso, are minority components, although their chain length distribution (C15-C27) is broad.

Experimental hepatic porphyria induced by oxadiazon in male mice and rats

Abstract Male mice and rats were fed a diet containing 1000 mg/kg of oxadiazon for 6 days. Fecal porphyrin was elevated in both mice and rats (from 30.2 to 1673.1 nmol/g dry wt and from 98.3 to 547.6 nmol/g dry wt, respectively). Porphyrin content of mouse bile was elevated from 5.7 to 439.0 nmol/g wet wt. Livers of treated animals contained mainly uroporphyrin, while protoporphyrin constituted the major porphyrin fraction in the feces of treated animals and in mouse bile. Urine of oxadiazon-treated rats contained coproporphyrin and uroporphyrin; total urine porphyrin content was 0.4 nmol/ml for control and 15.2 nmol/ml for treated rats. Porphobilinogen concentration was elevated in liver, kidneys, and blood plasma of treated animals. The activity of 5-aminolevulinic acid synthase was increased in the liver, but not in the kidneys of treated animals. Hepatic microsomal cytochrome P450 levels were decreased to about 50% of control values. The pattern of porphyrin accumulation and excretion in oxadiazon-induced experimental porphyria resembles the porphyrin pattern observed in human variegate porphyria.

Effect of erythropoietin on hepcidin expression in hemojuvelin-mutant mice

Transcription of the hepcidin (Hamp) gene is controlled by iron stores and the rate of erythropoiesis. Functional hierarchy between these two stimuli has not yet been completely established. It is also not known whether the erythropoiesis-related downregulation of Hamp expression utilises the bone morphogenetic protein/hemojuvelin (Bmp/Hjv) pathway. Hemojuvelin-mutant (Hjv-/-) mice treated with erythropoietin (EPO) at 50IU/mouse/day for three days displayed marked decrease in Hamp mRNA, demonstrating that hemojuvelin is not an indispensable component in EPO-induced Hamp gene downregulation. Irradiation of Hjv-/- mice prevented the EPO-induced decrease of Hamp mRNA, highlighting the role of erythropoiesis in Hamp gene regulation by EPO. After a single injection of EPO, Hamp mRNA levels were not significantly changed at 6h, but decreased at 10 and 24h. Chronic bleeding decreased hepatic Bmp6 mRNA levels; however, repeated EPO treatment did not change Bmp6 mRNA, suggesting that the erythropoietic regulator(s) act independently of the Bmp/Hjv pathway. Pretreatment of C57BL/6 mice with iron (5mg/mouse) almost completely inhibited the EPO-induced decrease of Hamp mRNA. This result suggests that administration of EPO to patients with transfusional iron overload is probably not associated with the risk of additional absorption of substantial amounts of iron from the diet.

Effect of tiagabine and topiramate on porphyrin metabolism in an in vivo model of porphyria

Administration of many antiepileptic drugs to patients with porphyria can precipitate an acute porphyric crisis. Information on the porphyrogenic activity of new antiepileptic drugs is still limited. In the presented study, the effects of tiagabine and topiramate on porphyrin metabolism were evaluated in an in vivo model of porphyria. Administration of the protoporphyrinogen oxidase inhibitor oxadiazon (12.5 mg/kg/day) for four days to male Wistar rats caused a partial block of porphyrin biosynthesis, thus mimicking the condition of quiescent variegate porphyria. Administration of phenobarbital (75 mg/kg/day) to oxadiazon-pretreated rats increased liver porphyrin content, liver porphobilinogen content (means 480 nmol/g, control less than 20 nmol/g) and urinary excretion of porphobilinogen (means 1,000 micromol/l, control less than 20 micromol/l). Tiagabine (75 mg/kg/day) and topiramate (75 mg/kg/day) increased liver porphobilinogen content (means 33 and 53 nmol/g respectively) and urinary porphobilinogen concentration (240 and 490 micromol/l respectively). Similar results were obtained in oxadiazon-treated BALB/c mice. In untreated rats, tiagabine and topiramate caused a moderate increase of hepatic pentoxyresorufin-O-dealkylase activity (approximately 100 and 200 pmol/min./mg respectively, controls 15 pmol/min./mg). These data demonstrate that administration of tiagabine or topiramate to oxadiazon-treated animals can provoke a condition resembling an acute porphyric attack and suggest that administration of these drugs to patients with suspected porphyria should be avoided. However, 5-day administration of both tiagabine and topiramate (75 mg/kg) is considerably less porphyrogenic than phenobarbital administered at the same dose.

Experimental hepatic uroporphyria induced by the diphenyl-ether herbicide fomesafen in male DBA/2 mice.

Hepatic uroporphyria can be readily induced by a variety of treatments in mice of the C57BL strains, whereas DBA/2 mice are almost completely resistant. However, feeding of the protoporphyrinogen oxidase-inhibiting herbicide fomesafen (0.25% in the diet for 18 weeks) induced hepatic uroporphyria in male DBA/2N mice (liver porphyrin content up to 150 nmol/g, control animals 1 nmol/g), whereas fomesafen-treated male C57BL/6N mice displayed only a slight elevation of liver porphyrins (approximately 5 nmol/g). The profile of accumulated hepatic porphyrins in fomesafen-treated DBA/2N mice resembled the well-characterised uroporphyria induced by polyhalogenated aromatic hydrocarbons, while histological examination confirmed the presence of uroporphyria-specific cytoplasmic inclusions in the hepatocytes. Uroporphyrinogen decarboxylase activity decreased to about 30% of control values in fomesafen-treated DBA/2N mice; microsomal methoxyresorufin O-dealkylase activity was slightly reduced. The amount of CYP1A1 and CYP1A2 mRNA, as determined by real-time PCR, was not significantly changed; mRNA encoding the housekeeping 5-aminolevulinic acid synthase was elevated 10-fold. Total liver iron was slightly increased. A similar uroporphyria was induced by the herbicide formulation Blazer, containing a structurally related herbicide acifluorfen, when fed to DBA/2N mice at a dose corresponding to 0.25% of acifluorfen in the diet. Since DBA/2 mice are almost completely resistant to all well-characterised porphyrogenic chemicals, the results suggest the possible existence of a yet unknown mechanism of uroporphyria induction, to which the DBA/2 mouse strain is more sensitive than the C57BL strain.

Effect of Erythropoietin, Iron Deficiency and Iron Overload on Liver Matriptase-2 (TMPRSS6) Protein Content in Mice and Rats

Matriptase-2 (TMPRSS6) is an important negative regulator of hepcidin expression; however, the effects of iron overload or accelerated erythropoiesis on liver TMPRSS6 protein content in vivo are largely unknown. We determined TMPRSS6 protein content in plasma membrane-enriched fractions of liver homogenates by immunoblotting, using a commercial antibody raised against the catalytic domain of TMPRSS6. Plasma membrane-enriched fractions were obtained by centrifugation at 3000 g and washing. TMPRSS6 was detected in the 3000 g fraction as a 120 kDa full-length protein in both mice and rats. Feeding of iron-deficient diet as well as erythropoietin treatment increased TMPRSS6 protein content in rats and mice by a posttranscriptional mechanism; the increase in TMPRSS6 protein by erythropoietin was also observed in Bmp6-mutant mice. Administration of high doses of iron to mice (200, 350 and 700 mg/kg) decreased TMPRSS6 protein content. Hemojuvelin was detected in the plasma membrane-enriched fractions of control animals as a full length protein of approximately 52 kDa; in iron deficient animals, the full length protein was partially cleaved at the N-terminus, resulting in an additional weak band of approximately 47 kDa. In livers from hemojuvelin-mutant mice, TMPRSS6 protein content was strongly decreased, suggesting that intact hemojuvelin is necessary for stable TMPRSS6 expression in the membrane. Overall, the results demonstrate posttranscriptional regulation of liver TMPRSS6 protein by iron status and erythropoietin administration, and provide support for the interaction of TMPRSS6 and hemojuvelin proteins in vivo.