Plasmids Shaped the Recent Emergence of the Major Nosocomial Pathogen Enterococcus faecium.

Enterococcus faecium is a gut commensal of humans and animals but is also listed on the WHO global priority list of multidrug-resistant pathogens. Many of its antibiotic resistance traits reside on plasmids and have the potential to be disseminated by horizontal gene transfer. Here, we present the first comprehensive population-wide analysis of the pan-plasmidome of a clinically important bacterium, by whole-genome sequence analysis of 1,644 isolates from hospital, commensal, and animal sources of E. faecium Long-read sequencing on a selection of isolates resulted in the completion of 305 plasmids that exhibited high levels of sequence modularity. We further investigated the entirety of all plasmids of each isolate (plasmidome) using a combination of short-read sequencing and machine-learning classifiers. Clustering of the plasmid sequences unraveled different E. faecium populations with a clear association with hospitalized patient isolates, suggesting different optimal configurations of plasmids in the hospital environment. The characterization of these populations allowed us to identify common mechanisms of plasmid stabilization such as toxin-antitoxin systems and genes exclusively present in particular plasmidome populations exemplified by copper resistance, phosphotransferase systems, or bacteriocin genes potentially involved in niche adaptation. Based on the distribution of k-mer distances between isolates, we concluded that plasmidomes rather than chromosomes are most informative for source specificity of E. faeciumIMPORTANCEEnterococcus faecium is one of the most frequent nosocomial pathogens of hospital-acquired infections. E. faecium has gained resistance against most commonly available antibiotics, most notably, against ampicillin, gentamicin, and vancomycin, which renders infections difficult to treat. Many antibiotic resistance traits, in particular, vancomycin resistance, can be encoded in autonomous and extrachromosomal elements called plasmids. These sequences can be disseminated to other isolates by horizontal gene transfer and confer novel mechanisms to source specificity. In our study, we elucidated the total plasmid content, referred to as the plasmidome, of 1,644 E. faecium isolates by using short- and long-read whole-genome technologies with the combination of a machine-learning classifier. This was fundamental to investigate the full collection of plasmid sequences present in our collection (pan-plasmidome) and to observe the potential transfer of plasmid sequences between E. faecium hosts. We observed that E. faecium isolates from hospitalized patients carried a larger number of plasmid sequences compared to that from other sources, and they elucidated different configurations of plasmidome populations in the hospital environment. We assessed the contribution of different genomic components and observed that plasmid sequences have the highest contribution to source specificity. Our study suggests that E. faecium plasmids are regulated by complex ecological constraints rather than physical interaction between hosts.

Chloropicrin-induced toxicity in the respiratory system.

Chloropicrin is a volatile and reactive chemical that has been utilized as a warfare agent and a pesticide to fumigate soil against insects, fungi and nematodes. It poses a health risk to humans and animals if inhaled. The main source of chloropicrin exposure is occupational and occurs during its manufacture, transport and fumigation. Chloropicrin is toxic via all routes of exposure but the main route of systemic exposure is inhalation of the ambient air. Thus, the toxicity mainly affects the respiratory system. After a low level exposure, the first sign is irritation of the upper respiratory tract and eyes. Irritation is mediated by the sensory nerve fibers, which coordinate further activation of various protective reflexes. Chloropicrin-induced irritation is generally reversible but can alter airway responsiveness to other inhalation toxicants. Severe exposures cause injuries in the respiratory tract, inflammation, and even life-threatening edema. Much of the chloropicrin-caused symptoms and toxicity in the respiratory system displays similarities with those evoked by chlorine, which is also a breakdown product of chloropicrin. This review summarizes the latest information on chloropicrin with emphasis on the toxicity in the respiratory system. The data indicates that oxidative stress, modification of macromolecules, mutations, dysfunctions of cell organelles and cell death are involved in acute chloropicrin-induced toxicity in the respiratory system.

European Surveillance System on Contact Allergies (ESSCA): results with the European baseline series, 2013/14.

BACKGROUND: Contact allergy is a common condition and can severely interfere with daily life or professional activities. Due to changes in exposures, such as introduction of new substances, new products or formulations and regulatory intervention, the spectrum of contact sensitization changes. OBJECTIVE: To evaluate the current spectrum of contact allergy to allergens present in the European baseline series (EBS) across Europe. METHODS: Retrospective analysis of data collected by the European Surveillance System on Contact Allergies (ESSCA, www.essca-dc.org) in consecutively patch-tested patients, 2013/14, in 46 departments in 12 European countries. RESULTS: Altogether, 31 689 patients were included in the analysis. Compared to a similar analysis in 2004, the prevalence of contact allergy to methylisothiazolinone went up to around 20% in several departments. In comparison, contact allergy to the metals nickel, cobalt and chromium remained largely stable, at 18.1%, 5.9% and 3.2%, respectively, similar to mostly unchanged prevalence with fragrance mix I, II and Myroxylon pereirae (balsam of Peru) at 7.3%, 3.8% and 5.3%, respectively. In the subgroup of departments diagnosing (mainly) patients with occupational contact dermatitis, the prevalence of work-related contact allergies such as epoxy resin or rubber additives was found to be increased, compared to general dermatology departments. CONCLUSION: Continuous surveillance of contact allergy based on network data offers the identification of time trends or persisting problems, and thus enables focussing in-depth research (subgroup analyses, exposure analysis) on areas where it is needed.

Occupational skin diseases: actual state analysis of patient management pathways in 28 European countries.

BACKGROUND: Work-related skin diseases (WSD) are caused or worsened by a professional activity. Occupational skin diseases (OSD) need to fulfil additional legal criteria which differ from country to country. OSD range amongst the five most frequently notified occupational diseases (musculoskeletal diseases, neurologic diseases, lung diseases, diseases of the sensory organs, skin diseases) in Europe. OBJECTIVE: To retrieve information and compare the current state of national frameworks and pathways to manage patients with occupational skin disease with regard to prevention, diagnosis, treatment and rehabilitation in different European countries. METHODS: A questionnaire-based survey of the current situation regarding OSD patient management pathways was carried out with experts on occupational dermatology and/or occupational medicine from 28 European countries contributing to the European Cooperation in Science and Technology (COST) Action TD 1206 (StanDerm) (www.standerm.eu). RESULTS: Besides a national health service or a statutory health insurance, most European member states implemented a second insurance scheme specifically geared at occupational diseases [insurance against occupational risks (synonyms: insurance against work accidents and occupational injuries; statutory social accident insurance)]. Legal standards for the assessment of occupationally triggered diseases with a genetic background differ between different countries, however, in most European member states recognition as OSD is possible. In one-third of the countries UV light-induced tumours can be recognized as OSD under specific conditions. CONCLUSION: OSD definitions vary between European countries and are not directly comparable, which hampers comparisons between statistics collected in different countries. Awareness of this fact and further efforts for standardization are necessary.

Chloropicrin induces endoplasmic reticulum stress in human retinal pigment epithelial cells.

Chloropicrin is an aliphatic volatile nitrate compound that is mainly used as a pesticide. It has several toxic effects in animals and can cause irritating and other health problems in exposed humans. Since the mode of chloropicrin action is poorly understood, the aim of this study was to investigate molecular responses underlying chloropicrin toxicity. We used human retinal pigment epithelial cells (ARPE-19) as a model cell type because the eyes are one of the main target organs affected by chloropicrin exposure. Transmission electron microscopy images revealed that exposure to a chloropicrin concentration that decreased cell viability by 50%, evoked the formation of numerous electron-lucent, non-autophagy vacuoles in the cytoplasm with dilatation of the endoplasmic reticulum (ER). Lower concentrations led to the appearance of more electron-dense vacuoles, which contained cytoplasmic material and were surrounded by a membrane resembling autophagy vacuoles. According to immunoblotting analyses chloropicrin increased the amount of the ER-stress related proteins, Bip (about 3-fold compared to the controls), IRE1α (2.5-fold) and Gadd 153/Chop (2.5-fold), evidence for accumulation of misfolded proteins in the ER. This property was further confirmed by the increase of reactive oxygen species (ROS) production (2-2.5-fold), induction of heme oxygenase-1 (about 6-fold), and increase in the level of the tumour suppressor protein p53 (2-fold). Thus, the cytotoxicity of chloropicrin in the retinal pigment epithelium is postulated to be associated with oxidative stress and perturbation of the ER functions, which are possibly among the mechanisms involved in oculotoxicity of chloropicrin.

Serum immunoglobulin A concentration in infancy, but not human milk immunoglobulin A, is associated with subsequent atopic manifestations in children and adolescents: a 20-year prospective follow-up study.

BACKGROUND: Serum and secretory IgA concentrations have been suggested to be inversely associated with allergic symptoms in children. Furthermore, low maternal milk IgA concentration has been suggested to be associated with the development of cow's milk allergy. OBJECTIVE: Our aim was to explore whether the serum IgA concentrations in infancy and the IgA concentration of maternal milk predict atopic manifestations in childhood and up to age 20 years. METHODS: A cohort of 200 unselected full-term newborns was prospectively followed up from birth to age 20 years with measurement of serum total IgA at ages 2 and 6 months. The mothers were encouraged to maintain exclusive breastfeeding for as long as possible. Total IgA concentration of maternal milk was measured at birth (colostrum, n=169) and at 2 (n=167) and 6 (n=119) months of lactation. The children were re-assessed at ages 5, 11 and 20 years for the occurrence of allergic symptoms, with skin prick testing and measurement of serum IgE. RESULTS: Children and adolescents with respiratory allergic symptoms and sensitization had a higher serum IgA concentration at age 2 months than the non-atopic subjects. Colostrum and breast milk IgA concentrations were not associated with the development of allergic symptoms in the recipient infant. However, maternal milk IgA concentration at 6 months of lactation was inversely associated with elevated serum total IgE and positive skin prick test to tree pollen in the offspring at age 20 years. CONCLUSIONS AND CLINICAL RELEVANCE: Increased serum IgA concentration at age 2 months is associated with the development of subsequent allergic symptoms and sensitization in childhood and adolescence. Maternal milk IgA concentrations are not associated with subsequent allergic symptoms in the recipient infant. The present study provides novel information on the role of IgA in the development of respiratory allergy and sensitization.

Serum cholesterol level in infancy is inversely associated with subsequent allergy in children and adolescents. A 20-year follow-up study.

BACKGROUND: Previous studies suggest an association between an altered lipoprotein profile and atopy. The association has been hypothesized to be due to alterations in the dietary fat intake, a factor possibly contributing to the increase of allergic diseases in industrialized countries. OBJECTIVE: We aimed at assessing whether there is an association between the serum lipid levels in infancy and subsequent development of allergic symptoms in childhood and adolescence. METHODS: A cohort of 200 unselected newborns was prospectively followed up from birth to age 20 years (from 1981 to 2002) with repeated measurements of total cholesterol from birth and throughout the first year of life. The subjects were re-examined at the ages of 5, 11 and 20 years, with assessment of the occurrence of allergic symptoms, skin prick testing (SPT) and measurement of total IgE and of the total, high- and low-density lipoprotein cholesterol. RESULTS: Children and adolescents with allergic symptoms, SPT positivity and an elevated IgE had lower total cholesterol levels in infancy and childhood than the non-atopic subjects. The difference was not detectable in cord blood, but became significant from age 2 months onward. CONCLUSION: The inverse association between the cholesterol level in infancy and subsequent manifestations of atopy seems not to be due to atopy-related dietary alterations, because it was already present in early infancy, when virtually all the infants were on a similar diet, i.e. on exclusive breastfeeding.

Retinol concentrations after birth are inversely associated with atopic manifestations in children and young adults.

BACKGROUND: Vitamin A has anti-inflammatory and immunomodulatory effects, and its deficiency results in impaired specific and innate immunity. Vitamin A is essential for inducing the gut-homing specificity on T cells. OBJECTIVE: As an impaired gut immune response in early infancy may contribute to the development of atopic sensitization, we looked for an association of plasma retinol concentrations and the subsequent development of allergic symptoms in healthy infants. METHODS: A cohort of 200 unselected, full-term newborns were followed up from birth to age 20 years. The plasma retinol concentration was determined in cord blood (n=97), at ages of 2, 4 and 12 months (n=95), and at ages 5 years (n=155) and 11 years (n=151). The subjects were re-examined at the ages of 5, 11 and 20 years with assessment of the occurrence of allergic symptoms during the preceding year, skin prick testing and measurement of serum total IgE. RESULTS: subjects with allergic symptoms or a positive skin prick test (SPT) in childhood or adolescence had lower retinol concentrations in infancy and childhood than symptom-free subjects. The difference was most pronounced at age 2 months. Retinol concentration at 2 months correlated inversely with positive SPT at ages of 5 and 20 years, and with allergic symptoms at age 20 years. CONCLUSION: Retinol concentration in young infants is inversely associated with the subsequent development of allergic symptoms. We propose that an inborn regulation of retinol may play a role in atopic sensitization, possibly through regulating the intestinal T cell responses.

Prolonged exclusive breastfeeding is associated with increased atopic dermatitis: a prospective follow-up study of unselected healthy newborns from birth to age 20 years.

BACKGROUND: Exclusive breastfeeding for the first 6 months is recommended by the World Health Organization and considered allergy preventive. However, it is not known whether prolonging exclusive breastfeeding for over 6 months provides further benefit in allergy prevention. OBJECTIVE: The aim of this prospective 20-year follow-up study was to find out whether the allergy protective effect can be enhanced by prolonging strictly exclusive breastfeeding for > or =9 months of age. A total of 200 unselected healthy newborns were enrolled in the study. Their mothers were encouraged to maintain exclusive breastfeeding for as long as possible. The number of infants on strictly exclusive breastfeeding was 167 at 2, 116 at 6, 36 at 9 and 7 at 12 months of age. Of the 200 infants, 42% had a family history of allergy. The children were re-assessed at ages 5 (n=163), 11 (n=150) and 20 years (n=164) with clinical examination, skin prick testing, and parental and personal structured interviews. RESULTS: Exclusive breastfeeding prolonged for > or =9 months was associated with atopic dermatitis (P=0.002) and symptoms of food hypersensitivity (P=0.02) at age 5 years, and with symptoms of food hypersensitivity at age 11 years (P=0.01), in children with a family history of allergy. CONCLUSION: Prolonging strictly exclusive breastfeeding for > or =9 months was not helpful in atopy prevention, instead, it was associated with increased atopic dermatitis and food hypersensitivity symptoms in childhood.

Comparison of two budesonide powder inhalers, Easyhaler and Turbuhaler, in steroid-naïve asthmatic patients.

The objective of this multicenter study was to compare the clinical efficacy, safety, and acceptability of Easyhaler and Turbuhaler for the delivery of budesonide 200 micrograms/dose twice daily in steroid-naïve asthmatic patients. Three hundred and twenty-six newly diagnosed, steroid-naïve adult patients with mild-to-moderate asthma were recruited into this randomized, double-blind, double-dummy, parallel-group study, comprising a 2-week run-in period and 8 weeks of treatment. Patients received budesonide inhalation powder 400 micrograms/day either via Easyhaler (n = 159) or via Turbuhaler (n = 167), plus salbutamol inhalation powder (100 micrograms/dose) via Easyhaler as rescue therapy. The study was completed by 292 patients: 143 in the Easyhaler group and 149 in the Turbuhaler group. The primary outcome variable, mean morning peak expiratory flow (PEF), improved significantly and almost similarly by 36.3 and 30.6 l/min, respectively, from run-in to weeks 7-8. At weeks 7-8, the mean (SE) difference in morning PEF between the two treatments was 7.1 (9.4) l/min (90% CI from -8.4 to 22.6) on per protocol analysis, which was within the defined limits for therapeutic equivalence. There were no significant differences between treatments in terms of secondary efficacy variables or adverse events. However, patients found Easyhaler more acceptable than Turbuhaler. The results show that budesonide via Easyhaler is clinically as effective as Pulmicort Turbuhaler when equal daily doses of budesonide are delivered to steroid-naïve asthmatic patients. Moreover, patients found Easyhaler more acceptable than Turbuhaler, and a majority would prefer Easyhaler if given a choice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters melatonin metabolism in fish hepatocytes.

Pineal hormone melatonin is an important regulator of endocrine and circadian rhythms in vertebrates. Since liver is assumed to be the major organ in the metabolism of this indole hormone, we investigated the effect of the known Ah-receptor agonist, 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) on melatonin metabolism in fish hepatocytes as well as the in vitro effect of melatonin on trout hepatic microsomal cytochrome P4501A (CYP1A) catalyst. Primary cell cultures of rainbow trout hepatocytes were exposed to [3H]melatonin (1 nM to 1 microM) alone and in combination with TCDD (50 pM) at 15 degrees C for 24 or 48 h. Analysis of melatonin and its metabolites in the culture medium and hepatocytes by HPLC revealed that about 96% of the added [3H]melatonin was metabolised after 24 h in both control and TCDD treated cultures. 3H-radioactivity was found mainly in the culture medium and less than 5% of the total 3H-radioactivity retained inside hepatocytes. Of the HPLC separated metabolites, one coeluted with 6-hydroxymelatonin and one unknown metabolite eluted after 6-hydroxymelatonin. In addition, two other metabolites were more water-soluble than 6-hydroxymelatonin and were considered to be conjugated products. Treatment of the hepatocytes with TCDD increased the amount of the major oxidated product, 6-hydroxymelatonin, about 2.5-fold after 24 h and 1.2-fold after 48 h exposure, respectively when compared with the control cultures. Whereas the amount of the unknown metabolite eluting after 6-hydroxymelatonin decreased about 1.3-fold after 24 h and 1.2-fold after 48 h exposure, respectively. Melatonin alone did not affect P4501A associated EROD-activity or CYP1AmRNA levels in the primary hepatocyte cultures. TCDD-treatment increased EROD-activity 3 to 5-fold and respective CYP1AmRNA content 6 to 14-fold, when compared with the control or melatonin-treated cultures. Furthermore, melatonin competitively inhibited EROD-activity in liver microsomes with a Ki value of 62.06+/-3.78 microM. The results show that TCDD alters metabolic degradation of melatonin in hepatocytes and suggest that P4501A may be an important P450 isoenzyme involved in oxidative metabolism of melatonin in fish liver.

Biochemical responses of fish sac fry and a primary cell culture of fish hepatocytes exposed to polychlorinated naphthalenes.

Chlorinated naphthalenes are planar halogenated aromatic compounds, which are widespread in the environment. Knowledge of their biochemical and toxicological actions in aquatic biota is, however, limited. The objective of this study was to assess the toxicity of highly chlorinated naphthalene congeners found in the aquatic environment on fish sac fry and to study their effects on xenobiotic metabolizing enzymes (CYP) using a short-term primary culture of fish hepatocytes and liver microsomes. A few days after hatching, rainbow trout sac fry were administered either Hallovax 1014, a mixture of 1,2,3,4,6,7-hexachloronaphthalene and 1,2,3,5,6, 7-hexachloronaphthalene (HxCN-mix), or 1,2,3,4,5,6, 7-heptachloronaphthalene (HpCN) (0.08, 0.8, and 4 microg/sac fry injected into the yolk sac). The exposure was terminated 2 weeks later. The naphthalene preparations did not cause any clinical signs of toxicity or difference in mortality rates between the control and treated groups. Immunohistochemical analysis of CYP1A expression in the treated sac fry revealed that staining was most pronounced in the hepatocytes and thereafter in kidney tubular epithelial cells. Moderate CYP1A staining was also seen in the mucosal epithelium of pyloric caecae and mild staining in the epithelium of olfactory organ. Staining in control sac fry was weak or absent. Exposure of the primary cell culture of trout hepatocytes to a low doses (

Assessment of PCBs and hydroxylated PCBs as potential xenoestrogens: In vitro studies based on MCF-7 cell proliferation and induction of vitellogenin in primary culture of rainbow trout hepatocytes.

In the present study, four structurally diverse polychlorinated biphenyls (PCBs) were chosen from a set of 20 PCBs selected to represent the 154 tetra- through hepta-chlorinated biphenyls. The purpose was to determine estrogenic activities of the chosen PCBs and five of their hydroxylated derivatives (OH-PCBs). A human breast cancer cell line (MCF-7) and primary cultures of rainbow trout (Oncorhyncus mykiss) hepatocytes were used to determine estrogenic effects. The PCBs 2,2',4,6,6'-pentachlorobiphenyl (104) and 2,2',3, 4', 5,6,6'-heptachlorobiphenyl (188), and the hydroxylated PCBs 2,2', 4',6'-tetrachloro-4-biphenylol (4'-50), 2',4', 6'-trichloro-4-biphenylol (4'-30), 2',3,5, 5'-tetrachloro-4-biphenylol (4'-72), 2',3,3',5', 6'-pentachloro-4-biphenylol (4'-112), and 2',3,4',5, 6'-pentachloro-4-biphenylol (4'-121) significantly increased MCF-7 cell proliferation. The coaddition of hydroxytamoxifen, an estrogen antagonist, inhibited increased cell proliferation. The activity of the hydroxylated PCBs 4'-50 and 4'-30 was significantly higher at all nominal concentrations tested as compared to the corresponding PCB, viz., PCB 104. The hydroxylated PCBs 4'-50, 4'-30, 4'-72 and 4'-112 induced vitellogenin synthesis in rainbow trout hepatocytes. Significant differences were found in the MCF-7 system between the parent PCB and its hydroxylated derivative, viz., for 4'-50/4'-30 and 104, and in the rainbow trout hepatocyte assay between 4'-112 and 112, respectively. No activity was observed for PCB 58 in any of the two assays in the present study. Even though cells from two different species (human and fish) are used in the present study, the results obtained by the two methods agree fairly well. In both studies the hydroxylated PCBs were more active than the PCBs, and 4'-30 was the most active compound second only to 17beta-estradiol. http://link.springer-ny. com/link/service/journals/00244/bibs/37n2p145.html

Effects of xenoestrogenic environmental pollutants on the proliferation of a human breast cancer cell line (MCF-7).

A human breast cancer cell line (MCF-7) was used to develop an in vitro screening assay for the detection of xenoestrogenic environmental pollutants. MCF-7 cells were cultured in DMEM containing 5% fetal bovine serum (FBS). An estrogenic response was defined as an increase in the frequency of proliferating MCF-7 cells, and was measured using a thymidine analog, bromodeoxyuridine, and flow cytometry. Di-2-ethylhexyl phthalate (DEHP) and 4-n-nonylphenol (4-n-NP) were used as model chemicals. The proliferation rate of S-phase cells after 24 h of exposure to various concentrations of 17beta-estradiol and to model compounds was compared with a positive and a negative control, containing 1 nM 17beta-estradiol and 0.1% ethanol, respectively. DEHP and 4-n-NP increased the frequency of proliferating MCF-7 cells in a dose-dependent manner. The lowest concentration that significantly increased the proliferation of MCF-7 cells was 10 microM for DEHP and 1 microM for 4-n-NP. The results showed that the assay is accurate and quick to perform. It may prove a valuable tool for screening potential estrogen-mimicking environmental pollutants.

Alkaline comet assay in rainbow trout hepatocytes.

The alkaline comet assay was performed to measure DNA integrity in fish hepatocytes. Primary cultures of rainbow trout hepatocytes were exposed to two known genotoxic compounds, hydrogen peroxide (H(2)O(2)) and benzo[a]pyrene (B[a]P), and to organic extracts of river sediments. The DNA damage in the form of single-strand breaks was monitored following the formation of DNA comets after alkaline electrophoresis. Exposure of the hepatocytes to H(2)O(2) for 2 hr increased strand breaks in a dose-related manner at the concentration range reported previously in studies with mammalian hepatocytes. B[a]P treatment led to a significant increase in strand breaks at the concentrations ranging from 0.1 to 10 muM after 4 hr of exposure. After 48 hr of exposure to B[a]P, the level of DNA strand breaks was lower than that of the control. The organic extracts obtained from river sediments significantly increased DNA strand breaks in trout hepatocytes, indicating the presence of genotoxic compounds in the sediment. The results show that the alkaline comet assay is a promising method by which to study the genotoxic potential of xenobiotics found in the aquatic environment.

Toxic effects of bleached and unbleached paper mill effluents in primary cultures of rainbow trout hepatocytes.

Toxic effects of unbleached (sulfate or sulfite) and bleached (sulfate) paper mill effluents were studied in a primary culture of rainbow trout liver cells. The effluents and control water from a clean area were extracted with diethyl ether and added to the cultures dissolved in dimethyl sulfoxide. Plasma membrane integrity was studied by measuring lactate dehydrogenase (LDH) leakage. The cellular content of glutathione (GSH) was used as an indicator of oxidative stress and the formation of reactive intermediates. Dose-response studies indicated that unbleached effluents contained more potent toxic substances than bleached effluents. Both unbleached and bleached effluents contained organic diethyl ether-extractable substances which increased cytochrome P450-dependent 7-ethoxyresorufin-O-deethylase (EROD) activities. The inducing effects were seen at concentrations substantially lower than those decreasing GSH content and increasing LDH leakage. Possible EROD inducing substances in bleached effluents are chlorinated organic compounds. Inducing compounds in unbleached effluents are yet to be identified. Furthermore, at higher concentrations the effluents contained substances that inhibited the cytochrome P450 system. The results show that the trout primary hepatocyte cultures afford a convenient in vitro method for screening cytochrome P450 inducing components extracted from industrial effluents to investigate mechanisms by which wastewaters cause injury in cells.