Polymorphisms in PAH metabolising enzyme CYP1A1 in colorectal cancer and their clinicopathological correlations.

CYP1A1 enzyme is integral to the biotransformation of polycyclic aromatic hydrocarbons to carcinogenic compounds. This study aimed to screen mutations in exon 7 (ex7) of CYP1A1 and investigate its clinicopathological correlations in fresh tissue samples from 85 patients (42 women; 43 men) with colorectal carcinoma (CRC). Tumour tissues and matched non-neoplastic mucosa tissues were collected prospectively. Genomic DNA was extracted from all tissues, and subject to high-resolution melt curve analysis for CYP1A1-ex7. Sanger sequencing was employed to detect specific mutations. Three known single nucleotide polymorphisms (SNPs) were identified in both tumour and matched non-neoplastic tissue for the same individual. Of the 85 patients, one third (n = 28) harboured either rs1048943, rs1799814, or rs41279188. Patients who had a SNP at ex7 of CYP1A1 were significantly more likely to be over 65 years of age (p = 0.015). Furthermore, individuals harbouring a SNP at exon7 showed a low incidence of perineural cancer infiltration (p = 0.025) when compared to the wild-type population. Overall, polymorphisms at exon 7 of CYP1A1 are present in patients with CRC and associated with a few clinicopathological characteristics.

UPLC-MS/MS analysis of CYP1A-mediated ethoxyresorufin-O-deethylation activity in the rat kidney microsomes.

Ethoxyresorufin (ER)-O-deethylation (EROD) activity has been widely used to assess cytochrome P450 1A (CYP1A) activity. The kinetics of CYP1A activity have been well characterized in the liver microsomes. However, studies in kidney microsomes are limited due to the much lower EROD activity in this organ. Here, we developed and validated a sensitive UPLC-MS/MS assay for the characterization of the EROD activity in the rat kidney microsomes. In a 50 µL reaction mixture, rat kidney microsomes (0.25 mg/mL) were incubated with ER (0.1-5 µM) and NADPH (1 mM) for 10 min. Acidic solvents, such as trichloroacetic acid or formic acid, used for quenching of the metabolic reactions and precipitation of the proteins, unexpectedly caused a spontaneous formation of resorufin (RES) from ER. Therefore, the metabolic reactions were terminated by adding acetonitrile, containing a deuterated internal standard (IS). Chromatographic separation was achieved on a C18 UPLC column, and the MS/MS ion transitions were 213.9/185.9 for RES and 220.0/192.0 for IS. The assay was validated in the linear range of 0.5 nM to 75 nM of RES and had a lower limit of quantitation of 0.5 nM. The overall recoveries of RES (90%-99%) and IS (85%-103%) were relatively high, with minimal matrix effect. The assay was successfully applied to the estimation of the Michaelis-Menten (MM) kinetics of EROD activity in the rat kidney microsomes (n = 3), which showed a maximum velocity of 2.68 ± 0.17 pmol/min/mg and a MM constant of 1.72 ± 0.24 µM (mean ± SD). It is concluded that our sensitive and specific analytical method, coupled with the optimized microsomal incubation conditions, provides a robust platform for further investigations of the effects of xenobiotics, environmental factors, or pathophysiologic conditions on the kinetics of EROD activity in the kidney microsomes.

A highly selective ratiometric fluorescent probe based on naphthalimide for detection and imaging of CYP1A1 in living cells and zebrafish.

Real-time monitoring of the cytochrome P450 1A1 (CYP1A1) activity in complex biological systems via a practical tool is highly sought after because of its significant role in the metabolism and bioactivation of various xenobiotics. Herein, according to slight differences in the 3D structure and substrate preference between CYP1A1 and its homologous CYP1A2, a series of novel ratiometric fluorescent probes were designed and synthesized using 1,8-naphthalimide because of its trait of naked-eye visualization and ratiometric fluorescence to achieve the detection of CYP1A1 in biological samples. Among these probes, NEiPN showed good water solubility, highly isoform selectivity and great sensitivity (LOD = 0.04874 nM) for CYP1A1 under simulated physiological conditions, which makes it favorable for monitoring CYP1A1 in vivo. Remarkably, NEiPN exhibited excellent reproducibility when it was used to detect the CYP1A1 content in human liver microsomes, which indicated that it has a great potential for quantifying the CYP1A1 content in real biological samples. Furthermore, NEiPN showed relatively low cytotoxicity and has been successfully applied in biological imaging in living cells and zebrafish. These findings indicate that NEiPN is capable of real-time monitoring of the activity of endogenous CYP1A1, which could provide support for CYP1A1-associated pathological processes.

Highly Variable Expression of CYP1A1 in Human Liver and Impact on Pharmacokinetics of Riociguat and Granisetron in Humans.

Cytochrome P450s (CYPs), a superfamily of enzymes, are involved in the biotransformation of endogenous and xenobiotic chemicals and mainly responsible for the metabolic clearance of widely prescribed drugs. Out of the 57 human isoforms, only a few, most notably CYP3A4, are considered to be important in this process. CYP1A1, one of the three isoforms of the CYP1 family, is widely believed to play an important role in the metabolism and activation of numerous procarcinogens, e.g., polyaromatic hydrocarbons (PAHs) or aromatic amines. It is also known that CYP1A1 is highly inducible by endogenous and exogenous factors, e.g., PAHs. However, CYP1A1 has not been considered to play a significant role in the metabolic clearance of drugs, since this isoform has been detected only in extrahepatic tissues in small amounts. In contrast to conventional wisdom, we herein demonstrate the expression of CYP1A1 protein in human liver microsomal preparations. The expression levels of CYP1A1 were quantified by Western blot and LC/MS analyses and corresponded well with enzymatic activities of highly selective CYP1A1 reactions. In a panel of 29 individual liver microsomal preparations, highly variable and substantial expression levels (up to ∼10 pmol/mg) were measured. Together with the high selectivity and especially the high metabolic efficiency of CYP1A1 shown for granisetron and riociguat, it is demonstrated that CYP1A1 plays an important role in the metabolic clearance of these drugs and is responsible for the clinically observed interindividual variability in their pharmacokinetics. Therefore, the importance of CYP1A1 in drug discovery and development needs to be reconsidered.

Activation and overexpression of the aryl hydrocarbon receptor contribute to cutaneous squamous cell carcinomas: an immunohistochemical study.

BACKGROUND: In vitro studies showed that the aryl hydrocarbon receptor (AHR) contributed to the development of cutaneous squamous cell carcinomas, but supporting clinical data are lacking. METHODS: Immunohistochemical analysis was used to detect the expression of AHR, CYP1A1, EGFR, and Ki-67 in 10 actinic keratosis (AK) cases, 10 Bowen disease (BD) cases, 20 cutaneous squamous cell carcinoma (cSCC) cases and 20 normal skin samples. H-scores were used to assess the immunoreactivity. RESULTS: Weak positive AHR immunoreactivity was found in all normal skin samples, while strong positive AHR immunoreactivity was found in atypical squamous proliferation (AK, BD and cSCC) cases. H-scores and the rate of strong immunostaining of the atypical squamous proliferation cases were higher than those of normal controls (p < 0.01). Nuclear expression of AHR was higher in atypical squamous proliferation cases than in normal controls (p < 0.01). H-scores and the nuclear expression rate of AHR were significantly higher in AK and BD cases than cSCC cases (p < 0.01). CYP1A1 expression was low and showed no differences among the four studied groups (p > 0.05). The H-score of AHR was positively correlated with EGFR expression (r = 0.54, p < 0.01) in atypical squamous proliferation cases but was not correlated with CYP1A1 (r = - 0.17, p = 0.295) and Ki-67 (r = - 0.48, p = 0.222) expression. CONCLUSION: AHR plays a vital role in cSCC pathogenesis. The overexpression and activation of AHR are involved in the early development of skin cancers. AHR expression correlates with EGFR expression and may influence cell proliferation. AHR is a valuable therapeutic target for skin cancers.

Synthesis of a selective ratiometric fluorescent probe based on Naphthalimide and its application in human cytochrome P450 1A.

Cytochrome P450s have brought considerable attention to researchers for their significant correlations with metabolic behaviors of procarcinogenic chemicals. To better understand the roles of CYP1A in biological and physiological systems, we developed a novel ratiometric fluorescence probe N-((2-hydroxyl ethoxy) ethyl)- 4-methoxy-1, 8-naphthalimide (NEMN) allowing for selectively and sensitively monitoring the target enzymes under physiological conditions and living cells. The probe was designed based on substrate predilection of CYP1A and its outstanding O-dealkylation capacity, and 1, 8-naphthalimide was chosen as fluorophore on account of its desirable photophysical properties. Absorption and emission spectra of the probe solution and reacted metabolism showed obvious red-shift with remarkable colour changes, which indicated that NEMN could be a promising ratiometric detector of CYP1A. Additionally, the selectivity assays displayed that NEMN only sensitive to CYP1A1 and CYP1A2 enzymes with scarce interference of other CYPs. Furthermore, the excellent linear relationships between the ratio of fluorescent intensities and incubation time and enzymes concentration signified time- and concentration- dependence of the probe, which were of desire benefit to quantify and monitor the CYP1A-involved biological behaviors in physiological conditions. The assay in real living samples (Human liver microsomes) further proved the analytical utility of the probe. Finally, the cytotoxicity assay and confocal fluorescence imaging demonstrated that this probe was of great promise for detecting the activity of endogenous CYP1A in human living cells.

Assessment of cytotoxicity, genotoxicity and 7-ethoxyresorufin-O-deethylase (EROD) induction in sediment extracts from New Zealand urban estuaries.

Sediments represent a major sink for contaminants resulting from industrial and agricultural activities - especially lipophilic substances. This study exclusively used in vitro methodologies to characterize specific toxicity effects of contaminants in sediment extracts from two urban New Zealand estuaries. Sediment extracts were prepared and tested for a range of biological endpoints. The micronucleus and comet assays in V79 cells were used to assess genotoxicity. Induction of 7-ethoxyresorufin-O-deethylase in piscine RTL-W1 cells was determined to estimate dioxin-like toxicity. Cytotoxic potentials were analyzed by neutral red uptake and MTT reduction. There was evidence of strong dioxin-like toxicity and moderate cytotoxicity. Genotoxicity was distinct in the micronucleus assay, but low in the comet assay. The results indicate the presence of chemicals in the sediments with the potential to pose a risk through multiple mechanisms of toxicity, the identities and amounts of which will be disclosed in a parallel study alongside with in vivo toxicity data.

Genistein decreases basal hepatic cytochrome P450 1A1 protein expression and activity in Swiss Webster mice.

Soy consumption has been associated with risk reduction for chronic diseases such as cancer. One proposed mechanism for cancer prevention by soy is through decreasing cytochrome P450 1A1 (Cyp1a1) activity. However, it is not known with certainty which soy components modulate Cyp1a1, or the characteristics or mechanisms involved in the responses after short-term (<20 days) dietary treatment without concomitant carcinogen-mediated induction. Therefore, the objective was to test the hypothesis that physiologic concentrations of dietary genistein and/or daidzein will decrease basal hepatic Cyp1a1 protein expression and activity in male and female Swiss Webster mice via inhibiting the bindings of aryl hydrocarbon receptor (AhR)-AhR nuclear translocator (ARNT) and estrogen receptor-α to the Cyp1a1 promoter region xenobiotic response element. The mice were fed the AIN-93G diet supplemented with 1500 mg/kg of genistein or daidzein for up to 1 week. Genistein, but not daidzein, significantly decreased basal hepatic microsomal Cyp1a1 protein expression and activity. AhR protein expression was not altered. Molecular mechanisms were investigated in Hepa-1c1c7 cells treated with 5 µmol/L purified aglycones genistein, daidzein, or equol. Cells treated with genistein exhibited inhibitions in ARNT and estrogen receptor-α bindings to the Cyp1a1 promoter region. This study demonstrated that genistein consumption reduced constitutive hepatic Cyp1a1 protein expression and activity, thereby contributing to the understanding of how soy isoflavone aglycones modulate cytochrome P450 biotransformation enzymes.

Differential effects of metal ions on TCDD-induced cytotoxicity and cytochrome P4501A1 gene expression in a zebrafish liver (ZFL) cell-line.

Trace metal ions and trace organic compounds are common co-contaminants in the environment that pose risks to human health. We evaluated the effects of four metal ions (As(3+), Cu(2+), Hg(2+), and Zn(2+)) on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced cytotoxicity and the expression of the cytochrome P4501A1 gene (cyp1a1) in the zebrafish liver (ZFL) cell line. A metal accumulation study showed that Cu and Zn did not accumulate in ZFL cells. However, As and Hg did accumulate, which resulted in the inhibition of TCDD-mediated induction of cyp1a1 mRNA and protein expression, and 7-ethoxyresorufin O-deethylase activity. A luciferase assay showed that both As(3+) and Hg(2+) inhibited the TCDD-induced activity of gene constructs containing either synthetic 3XRE or a distal cyp1a1 promoter region, implying that the decreased levels of TCDD-induced cyp1a1 were due to transcriptional effects. A proteomic study showed that the toxic effects of As(3+) might be due to changes in cellular metabolic processes, the cellular stimulation response and the cellular redox state in ZFL cells.

A Highly Selective Ratiometric Two-Photon Fluorescent Probe for Human Cytochrome P450 1A.

Cytochrome P450 1A (CYP1A), one of the most important phase I drug-metabolizing enzymes in humans, plays a crucial role in the metabolic activation of procarcinogenic compounds to their ultimate carcinogens. Herein, we reported the development of a ratiometric two-photon fluorescent probe NCMN that allowed for selective and sensitive detection of CYP1A for the first time. The probe was designed on the basis of substrate preference of CYP1A and its high capacity for O-dealkylation, while 1,8-naphthalimide was selected as fluorophore because of its two-photon absorption properties. To achieve a highly selective probe for CYP1A, a series of 1,8-naphthalimide derivatives were synthesized and used to explore the potential structure-selectivity relationship, by using a panel of human CYP isoforms for selectivity screening. After screening and optimization, NCMN displayed the best combination of selectivity, sensitivity and ratiometric fluorescence response following CYP1A-catalyzed O-demetylation. Furthermore, the probe can be used to real-time monitor the enzyme activity of CYP1A in complex biological systems, and it has the potential for rapid screening of CYP1A modulators using tissue preparation as enzyme sources. NCMN has also been successfully used for two-photon imaging of intracellular CYP1A in living cells and tissues, and showed high ratiometric imaging resolution and deep-tissue imaging depth. In summary, a two-photon excited ratiometric fluorescent probe NCMN has been developed and well-characterized for sensitive and selective detection of CYP1A, which holds great promise for bioimaging of endogenous CYP1A in living cells and for further investigation on CYP1A associated biological functions in complex biological systems.

Flaxseed reduces the pro-carcinogenic micro-environment in the ovaries of normal hens by altering the PG and oestrogen pathways in a dose-dependent manner.

The objective of the present study was to find the optimum dose of flaxseed that would decrease PG and alter oestrogen pathway endpoints implicated in ovarian cancer. In the study, four groups of fifty 1.5-year-old chickens were fed different amounts of flaxseed (0, 5, 10 or 15% of their total diet) for 4 months and were then killed to collect blood and tissues. Levels of flaxseed lignan metabolites, Enterolactone (EL) and Enterodiol (ED) were measured in the serum, liver and ovaries by liquid chromatography-MS/MS, and n-3 and n-6 fatty acid (FA) levels were measured by GC. The effects of the varied flaxseed doses were assessed by measuring levels of PGE2 and oestrogen metabolites (16-hydroxyestrone (16-OHE1) and 2-hydroxyestrone (2-OHE1)) as well as by analysing the expression of the oestradiol metabolising enzymes CYP3A4 (cytochrome p450, family 3, subfamily A, polypeptide 4), CYP1B1 (cytochrome p450, family 1, subfamily B, polypeptide 1) and CYP1A1 (cytochrome p450, family 1, subfamily A, polypeptide 1) and that of oestrogen receptor α (ERα) in the ovaries. The ratio of n-3:n-FA increased with an increase in flaxseed supplementation and corresponded to a dose-dependent decrease in cyclo-oxygenase-2 protein and PGE2 levels. EL and ED increased in the serum, liver and ovaries with increased concentrations of flaxseed. Flaxseed decreased the expression of ERα in the ovaries. The ratio of 2-OHE1:16-OHE1 in the serum increased significantly in the 15% flaxseed diet, and there was a corresponding increase in CYP1A1 in the liver and decrease in CYP3A4 in the ovaries. CYP1B1 mRNA also decreased with flaxseed diet in the ovaries. The 15% flaxseed-supplemented diet significantly decreased inflammatory PGE2, ERα, CYP3A4, CYP1B1 and 16-OHE1, but it increased CYP1A1 and 2-OHE1, which thus reduced the inflammatory and pro-carcinogenic micro-environment of the ovaries.

Analysis of potential risk factors for cancer incidence in patients with aristolochic acid nephropathy from Wenzhou, China.

BACKGROUND: The purpose of this study was to investigate the cancer incidence in patients with end-stage aristolochic acid nephropathy (AAN). METHODS: A total of 102 patients with end-stage AAN treated in our hospital between 2004 and 2013 were included in this study. The correlation of cancer incidence with age, gender, dosage of aristolochic acid (AA), the type of renal replacement therapies, and the polymorphisms of quinone oxidoreductase 1 (NQO1) C609T and cytochrome P450 1A1 (CYP1A1) A4889G was examined. RESULTS: The cancer incidence rate in our patients was 41.2% (42 in 102) including 39 cases of urinary cancer. The mortality rate in the patients with cancer was significantly higher than that in the patients without cancer (31%, 13/42 vs. 11.7%, 7/60, p<0.05). Thirteen patients developed cancer before entering end-stage renal disease (ESRD). Cancer incidence was significantly associated with the dosage of AA consumption (p=0.091). Hemodialysis, peritoneal dialysis and renal transplant did not affect the cancer incidence in our patients differently, but appeared to be associated with cancer at particular locations of urinary system. The patients undergoing hemodialysis seemed to more likely have bladder cancer (72.72%), while the patients receiving peritoneal dialysis appeared to develop cancer predominantly in the upper urinary tract (66.67%). CONCLUSIONS: The cancer initiation in our patients seems significantly correlate with the dosage of AA consumption. Different renal replacement therapies appear to be associated with cancer at particular locations of urinary system in our patients.

The effect of CYP1A1 and GSTP1 isozymes on the occurrence of aortic aneurysms.

BACKGROUND: Aortic aneurysms are vascular diseases that are associated with high mortality and morbidity. Cytochrome P450 CYP1A1 and glutathione S-transferase (GSTP1) isozymes were searched and compared with the patients who had experienced aortic surgery due to aortic aneurysm and atherosclerotic patients without aneurysm to find the relation of the oxidative stress with the aneurysms. MATERIALS AND METHODS: Study group consisted of the patients with the diagnosis of aortic aneurysm (group I, n: 12) and control group who were operated for coronary bypass surgery: preoperatively drug users (group II, n: 21) and nonusers (group III, n: 15). Paraffin sections (4 µm thick) of aortic biopsy materials were stained with hematoxylin and eosine, CYP1A1 and GSTP1 immunohistochemical markers. The specimens were evaluated using light microscopy at 40- to 400-fold magnification. RESULTS: The expressions of CYP1A1 and GSTP1 isozymes were found statistically significantly higher in the patients who have an aortic aneurysm than both the control groups (p < 0.05). There was no significant association between protein expressions, drugs and duration of usage, patient's demographic variables, and smoking (p > 0.05). CONCLUSIONS: In this pioneering study, CYP1A1 and GSTP1 isozymes are related with the aneurysms. The strategy that prevents the oxidative stress for the patients who had aortic aneurysms could be a valuable choice of searching to effect the aneurysmal progression.

Biomarker responses in sunfish species and largemouth bass from the Saluda River, South Carolina.

The upstate and Piedmont region of South Carolina is a rapidly urbanizing area as a result of a steadily growing population. This increase in population and development has the potential to negatively impact local aquatic systems like the Saluda River due to increased pollution from runoff, and effluents from industrial and wastewater treatment facilities. During the summer months of 2010, 159 fish from the Centrarchidae family (sunfish species (Lepomis) and largemouth bass - Micropterus salmoides) were collected from 13 sites along the Saluda River. A suite of biomarker assays, including ethoxyresosufin-O-deethylase, bile fluorescence, glutathione S-transferase, thiobarbituric acid reactive substances, bile estrogens, acetylcholinesterase inhibition, metallothionein and tissue metal levels were applied to investigate the impacts of diminished water quality on fish health. Results indicate that fish from the Saluda River are responding to contamination in a site specific manner, with up to four significant biomarker responses in the most impacted sites. Sampling sites in the lower portion of the Saluda watershed are less impacted by pollution than the upper and central sections. The observed biomarker responses can be explained by the proximity of urban areas, point sources and general land use, and demonstrate the applicability of biomarkers in environmental biomonitoring programs.

Expression profile of CYP1A1 and CYP1B1 enzymes in endometrial tumors.

The cytochrome P450 CYP1A1 and CYP1B1 enzymes are phase I extrahepatic enzymes involved in the activation of pro-carcinogenic compounds to carcinogenic metabolites. Although differential overexpression of CYP1A1 and CYP1B1 has been documented at the messenger RNA (mRNA) and protein level, studies that have examined CYP1 expression by enzyme activity assays are limited. In the current study, the expression of CYP1A1 and CYP1B1 was investigated in a panel of human tumors of endometrial origin by quantitative reverse transcriptase PCR (qRT-PCR), Western blotting, and enzyme activity assays. The data revealed that approximately 36 % (5/14) and 43 % (6/14) of the endometrial tumors overexpressed CYP1A1 and CYP1B1 mRNA, whereas in 57 % of the endometrial tumors, CYP1 mRNA levels were downregulated. The mean mRNA levels of CYP1B1 and CYP1A1 in endometrial tumors did not show a significant difference compared to normal tissues (p > 0.05). Western blotting confirmed the qRT-PCR results and CYP1A1 and CYP1B1 proteins were shown to be downregulated in 7/14 (50 %) of the tumors and overexpressed in 4/14 (29 %) of the tumors. As regards to enzyme activity, 21 % (3/14) of the endometrial samples revealed elevated CYP1 activity levels across the tumor counterparts. Overall, the data suggest a putative downregulation of CYP1A1 and CYP1B1 expression in endometrial tumors, whereas overexpression of active CYP1 enzymes in 21 % of the tumors highlights the potential use of the latter enzymes as chemotherapeutic targets in endometrial cancer.

Repeated treatment with furazolidone induces multiple cytochrome p450-related activities in chicken liver, but not in rat liver.

The nitrofuran antimicrobial agent, furazolidone (FZ), is still used in veterinary medicine in some countries in the Middle and Far Eastern countries. The present study aimed to investigate the effects of successive bolus doses of FZ and its metabolite 3-amino-2-oxazolidinone (AOZ) on cytochrome P450 (CYP)-related activities in the livers of rats and chickens. Female Wistar rats and white Leghorn chickens were orally administered FZ once a day for 4 consecutive days. FZ-treated chickens showed an increase in multiple CYP-related activities, however, rats treated with FZ did not show these changes. In chickens, treatment with FZ also induced production of microsomal CYP2C6-like apoprotein. The present study demonstrated that FZ caused a multiple-type induction of CYP-related activities in chickens, but not in rats.

A simple and reliable in vivo EROD activity measurement in single Fundulus heteroclitus embryo and larva.

Dioxin-like compounds (DLC) induce toxic responses in early life stages of fish through activation of the aryl hydrocarbon receptor (AhR) which is frequently assessed by ethoxyresorufin-O-deethylase (EROD) activity. A novel spectrofluorimetric method was developed to quantitatively assess EROD activity in individual living embryos and prolarvae of a marine model fish species, the mummichog Fundulus heteroclitus. This in vivo method is based on the measurement of the production of resorufin by single live embryos or prolarvae after 5 h incubation with ethoxyresorufin. Freshly fertilized eggs were treated topically from 2.5 to 50 pg egg⁻¹, with 3,3',4,4',5-pentachlorobiphenyl (PCB126), a prototypical DLC. EROD activity was assessed in embryos (7 days post-fertilization) and prolarvae (16 days post-fertilization). Resorufin was measured both in the culture medium (25‰ seawater) and in whole fish homogenates, to assess the percentage retained in the body. Approximately 95% and 17% of the resorufin produced in vivo was retained in embryos and prolarvae respectively. EROD activity in homogenates of embryos and in the culture medium of prolarvae increased linearly with dose. EROD activity measured by the in vivo method was highly correlated to that measured by a traditional in vitro technique using S9 fractions for both embryos and prolarvae. Both in vivo and in vitro EROD activity were higher in prolarvae than in embryos pretreated with PCB126. EROD induction measured in prolarvae by the in vivo and in vitro methods were similar whereas higher induction was measured in vivo than in vitro in embryos. The in vivo method was more sensitive and as reliable as the in vitro technique, and required a lower number of fish (4 compared to 3 pools of 5). This in vivo method is useful to link EROD induction in individual embryos or prolarvae to other organism-level responses. Further studies with other categories of xenobiotics should be performed to assess potential toxic effects on resorufin absorption/excretion processes which could affect in vivo measurements.

Evaluation of EROD and CYP3A4 activities in earthworm Eisenia fetida as biomarkers for soil heavy metal contamination.

In this study, the effects of heavy metals (Cd, Cu, Pb, Zn) on EROD and CYP3A4 activities in the earthworm Eisenia fetida were evaluated to find out their possible induction and potential as biomarkers for soil heavy metal contamination. The earthworms were exposed to increasing concentrations of Cd (0.1-8 mg L(-1)), Cu (10-200 mg L(-1)), Pb (20-400 mg L(-1)) or Zn (50-400 mg L(-1)) in filter papers for 48 h. EROD activity was significantly changed in dose-dependent manners after exposure to each of the four metals. CYP3A4 activity was significantly induced by Cd and Pb, rather by Cu and Zn. This is the first report on heavy metal-induced changes of CYP3A4 activity in earthworms. Among the four heavy metals, Cd was the most potent inducing EROD and CYP3A4. While EROD and CYP3A4 activities showed a similar trend, EROD is more sensitive than CYP3A4 activity in E. fetida as a biomarker for heavy metals pollution.

Stock-specific variation of trophic position, diet and environmental stress markers in Atlantic salmon Salmo salar during feeding migrations in the Baltic Sea.

This study investigated stock-specific variation in selected ecophysiological variables during the feeding migrations of Atlantic salmon Salmo salar in the Baltic Sea. Oxidative stress biomarkers and EROD (ethoxyresorufin-O-deethylase, Cyp1A enzyme) activity were used as indicators of possible environmental stress and stable isotopes as determinants of diet and trophic position. Latvian S. salar stocks Daugava and Gauja had distinct stable-isotope signatures compared to the other stocks, indicating differences in migration patterns, residency or arrival times, or dietary specialization among stocks. Salmo salar originating from Daugava and Gauja also had lower catalase enzyme activity than the other stocks. Post-smolts originating from rivers of the Gulf of Finland had elevated EROD activities compared to fish of the same age from Bothnian Bay rivers, which could indicate exposure to organochlorine pollutants. No other stock-specific differences in oxidative stress biomarkers were found. The study demonstrates how genetic, oxidative stress biomarker, EROD and stable-isotope data may be combined to study trophic position, prey prevalence and environmental stress of mixed S. salar stocks foraging in the sea.

Liver ethoxyresorufin-O-deethylase and brain acetylcholinesterase in two freshwater fish species of South America; the effects of seasonal variability on study design for biomonitoring.

Responses at low levels of biological organization to evaluate environmental changes and water quality have been used for many years. South America is no different, and recently biochemical endpoints in fish have been used to assess the impacts of industrial and sewage effluents on wild fish populations. For Chilean native freshwater fish, basic biological data is scarce and data on 7-ethoxyresorufin-O-deethylase (EROD) and Acetylcholinesterase (AChE) activity is practically absent. Moreover, extensive variation in these two biochemical endpoints exists among species and seasons. In this article we evaluate seasonal variation in liver EROD and brain AChE activities in Trichomycterus areolatus and Percilia gillissi, two widely distributed native freshwater fish species in central Chile. We observed a marked seasonality in hepatic EROD activity in both species, with maximums for P. gillissi during winter months and sex differences in February, July, August and December. T. areolatus showed no sex differences, and peaks in EROD activity in the middle of summer, winter and late spring. Species differences in EROD activity were observed with activity being 1-2 orders of magnitude higher in P. gillissi compared to T. areolatus. Scarce seasonal variation and no sex related differences in brain AChE for both species were observed. Multivariate analysis (PCA) indicated that physical water quality parameters had some degree of responsibility for the seasonal responses found. The seasonal variability data of these biochemical endpoints were used to optimize study design for future monitoring programs, planning timing of sampling, increasing statistical power by collecting specific sample sizes required.