An interlaboratory study on the use of steroid hormones in examining endocrine disruption.

In recent years, there has been an increased use of the measurement of sex steroid hormone levels in the blood of animals exposed to chemicals as an indicator of reproductive impairment or an alteration in endocrine function. Although levels of hormones are often compared among animals and laboratories, there has been no study to examine the between-laboratory variability in actual steroid measurements. Therefore, we initiated a study with white sucker collected from a site receiving pulp mill effluent, previously documented as having reduced steroid levels, to address this issue. Samples of plasma and media from in vitro gonadal incubations were delivered to eight outside laboratories with the ability to measure steroid hormones. These laboratories ranged from well-established fish endocrine laboratories to wildlife toxicology laboratories, which have recently implemented the methods to measure steroid hormones. In this study, we have considered both the absolute measure of steroid content between laboratories as well as the ability to discriminate between reference and exposed populations as important criteria when evaluating the utility of these measures. Of the eight outside laboratories conducting the analyses, six detected identical site differences in circulating levels of testosterone and 17beta-estradiol to those documented by our Burlington laboratory (ON, Canada). However, the absolute value of the steroid hormones measured in the plasma varied significantly (plasma testosterone 0.6-23.1 ng/ml, 17beta-estradiol 77.6-1782.7 pg/ml) with coefficients of variation of 70.4% and 60.3% respectively. Similar results were demonstrated for the measurement of steroid hormones in media following in vitro gonadal incubation. Although there was a fair amount of variability in the absolute measure of steroid hormone levels, we would predict a far greater coherence of interlaboratory results through the sharing of reagents and the use of a common methodology between laboratories. These results are very promising, providing evidence for the inclusion of steroid hormones in monitoring endocrine disruption in wildlife species.

Determination of heavy metals and pesticides in ginseng products.

Medicinal plants may carry residuals of environmentally persistent pesticides or assimilate heavy metals in varying degrees. Several factors may influence contaminant accumulation, including species, level and duration of contaminant exposure, and topography. As part of a program for assessment of the quality of herbal medicines, we have analyzed 21 over-the-counter ginseng (Panax ginseng) products in various dosage forms. Chromium, mercury, and arsenic were undetectable above their limits of detection in both liquid and solid samples; while cadmium, lead, and nickel were present in the majority of samples. The chlorinated pesticide levels varied widely. In most samples, the total concentration of pesticides was below 100 ppb; while in 5 samples the total concentration exceeded 100 ppb.

Elevation of serum 17-beta-estradiol in channel catfish following injection of 17-beta-estradiol, ethynyl estradiol, estrone, estriol and estradiol-17-beta-glucuronide.

17-beta-Estradiol is naturally converted in numerous organisms to various derivatives/metabolites, which may be excreted from the organism into its immediate external environment. There is a paucity of data regarding the biological effects of these derivatives/metabolites on aquatic organisms. Male channel catfish (200-500 g, N=5, 12-18 months) were injected with 1 mg/kg 17-beta-estradiol (E2), ethynyl estradiol (EE2), estrone, estriol or E2-17-beta-glucuronide with subsequent measurements of vitellogenin (Vtg) and serum E2 concentrations 7 days post injection. EE2 and E2 gave the largest magnitude of Vtg response followed by estrone and estriol. Exposure to EE2, estrone, and E2-17-beta-glucuronide all induced significant increases in serum E2 concentrations. This study indicates that metabolites of E2 are also estrogenic and may potentially disrupt estrogen feedback loops within aquatic organisms.

Mercury in sediment and fish from North Mississippi Lakes.

Sediments and/or fish were collected from Sardis, Enid and Grenada Lakes, which are located in three different watersheds in North Mississippi, in order to assess mercury contamination. The mean total mercury concentration in sediments from Enid Lake in 1997 was 0.154 mg Hg/kg, while 1998 sediment concentrations in Sardis, Enid and Grenada Lakes were 0.112, 0.088 and 0.133 mg Hg/kg, respectively. Sediment mercury concentrations in 1999 were similar in all three lakes but, generally lower than 1998. Mean total mercury concentrations in edible fillets of fish collected from Enid Lake in 1998 were above the human health FDA action level (> 1.0 mg Hg/kg) for bass (1.40), crappie (1.69) and gar (1.89); however, tissue concentrations were less than 1.0 mg Hg/kg in carp (0.63) and catfish (0.82). Human hazard indexes for each species was > or = 1 for both adults and children, indicating that there is a potential for toxic effects to occur. In addition, calculated consumption limits indicate that adults may consume 4-12 oz. of fish per month, depending on the species consumed. For children, 2 oz. per month may be consumed. Further studies are needed to determine the exact environmental consequences and human health impacts associated with mercury contamination in North Mississippi and the Southeastern United States.

Toxicokinetic interactions and survival of Hyalella azteca exposed to binary mixtures of chlorpyrifos, dieldrin, and methyl mercury.

Chemical mixture interactions of chlorpyrifos, dieldrin, and methyl mercury were evaluated in Hyalella azteca. Survival of adult and juvenile organisms was evaluated following exposure to individual chemicals and in binary combinations. Binary interactions of the model chemicals on survival of adult and juvenile H. azteca were evaluated by concentration-response curve analysis as additive, synergistic, antagonistic, or independent. Chlorpyrifos and methyl mercury interacted additively, while dieldrin interacted independently with both chlorpyrifos and methyl mercury. Toxicodynamic interactions were evaluated by measuring accumulation and elimination of each model toxicant in the presence of a second toxicant. Chlorpyrifos significantly increased the amount of methyl mercury accumulated in the first 12 h of exposure. Dieldrin did not interact with chlorpyrifos or methyl mercury in the accumulation or elimination experiments. Accumulation of chlorpyrifos was not observed. Results of the current investigation demonstrate that chlorpyrifos and methyl mercury interact additively, which would otherwise be predicted to act independently.

Interactions of chlorpyrifos and methyl mercury: a mechanistic approach to assess chemical mixtures.

The mechanism of interaction between chlorpyrifos, an organophosphate insecticide, and methyl mercury was assessed utilizing the amphipod, Hyalella azteca. Previous studies have demonstrated that chlorpyrifos and methyl mercury interact additively with survival as the endpoint. In addition, exposure to chlorpyrifos and methyl mercury increased the accumulation and decreased the elimination of methyl mercury. To further examine the mechanism responsible for these interactions, the in vivo and in vitro inhibition of acetylcholinesterase enzyme activity was assessed following exposure to methyl mercury and chlorpyrifos. In vivo, methyl mercury antagonized the effects of chlorpyrifos on acetylcholinesterase inhibition. Additional studies demonstrated that methyl mercury did not affect the in vitro bioactivation of chlorpyrifos or the subsequent inhibition of acetylcholinesterase enzyme activity. Chemical-chemical interactions were examined utilizing chromatographic techniques and suggested the formation of a chlorpyrifos-methyl mercury complex. The formation of this complex may result in increased accumulation of methyl mercury, apparent additive toxicity, and protection against chlorpyrifos-mediated acetylcholinesterase inhibition.

Exposure to environmentally relevant concentrations of different nonylphenol formulations in Japanese medaka.

The time course of exposure to p-nonylphenol (NP) from two different sources was compared to equivalent exposures of 17-beta-estradiol (E2) and a solvent control (ethanol; EtOH). Japanese medaka were exposed for 4 days to a nominal concentration of 20 micrograms/l of either NP-I (Schenectady International, Inc.), NP-II (Aldrich), or E2, and were then placed in untreated water for 5 days. Tissue samples were taken at two time points during the 4-day exposure and two time points during the 5 days following exposure. Liver homogenates were analyzed using a western blot to detect vitellogenin (VTG) and quantified by measuring the optical density for each lane. Preliminary results indicate that E2 significantly increased VTG staining above the level observed in EtOH-treated controls for both males and females. A two-way analysis of variance (ANOVA) indicates that NP from both sources, as well as E2, significantly increased VTG staining in males (ANOVA, n = 48, P < 0.001; Tukey pairwise tests, all P < 0.008). A significant increase in VTG was observed in E2-treated males and females the first day following transfer into toxicant-free water (two-way ANOVAs, both n = 48, P < 0.003; Tukey pairwise tests, all P < 0.019). If confirmed, this extended response observed for low-level exposures may represent a significant factor for sampling scenarios following pulsitile exposure.

Developmental evaluation of a potential non-steroidal estrogen: triclosan.

Triclosan is an antibacterial agent commonly used in industry and often detected in waste-water effluent. The potential of triclosan to act as an endocrine disruptor was examined because its chemical structure closely resembles known non-steroidal estrogens (e.g. DES, bisphenol A). Japanese medaka fry (Oryzias latipes) were exposed for 14 days beginning 2 days post-hatch to triclosan (100, 10, 1 micrograms/l), 17-beta estradiol (E2; 1 microgram/l), or a solvent control (ethanol). Two months post-exposure, the phenotypic sex of each adult was assessed visually using sexually dimorphic fin shape and size. The proportion of females in each group was similar for triclosan-exposed animals and solvent-treated controls (ethanol 53%, 1 ppb 58%, 10 ppb 45%, 100 ppb 36%) although E2 treatment did produce 92% female adults. Sexually dimorphic fin traits were quantified to look for potential effects of triclosan and E2 on the development of secondary sexual characters. These results do not support the hypothesis that triclosan is potently estrogenic. However, changes in fin length and non-significant trends in sex ratio suggest triclosan is potentially weakly androgenic.

Comparative vitellogenic responses in three teleost species: extrapolation to in situ field studies.

Induction of vitellogenin (VTG) was compared among three teleostean species to determine their relative sensitivity of exposure to 17 beta-estradiol (E2). Japanese medaka (Oryzias latipes), sunshine bass (Morone saxatalis x Morone chrysops) and channel catfish (Ictalurus punctatus) were exposed to aqueous concentrations of E2 ranging from 10 to 100,000 ng/l for 21 days. Respective EC50 values for plasma VTG detected by western blot in medaka, catfish and bass were 200, 170 and 1560 ng E2/l. Since these EC50 values are based on VTG induction curves calculated relative to control values, they indicate differences in species' sensitivity to E2 exposure. Catfish and bass VTG responses obtained in laboratory exposures were compared to VTG responses previously observed with 21-day wastewater treatment plant effluent exposures. Plasma VTG induction in effluent-exposed fish ranged from 14 to 82% above reference values depending on species. Extrapolation of field responses with laboratory-exposed fish indicate catfish and bass were exposed to the equivalent of 27-240 ng E2/l in sewage effluent.

Toxicological interactions of chlorpyrifos and methyl mercury in the amphipod, Hyalella azteca.

The mechanism of interaction between chlorpyrifos, an organophosphate insecticide, and methyl mercury, an organometal, was assessed utilizing the amphipod, Hyalella azteca. Previous studies have demonstrated that chlorpyrifos and methyl mercury interact additively, with survival as the endpoint. In addition, exposure to chlorpyrifos and methyl mercury increased the accumulation and decreased the elimination of methyl mercury. To elucidate the mechanism responsible for these interactions, biochemical mechanisms indicative of chlorpyrifos and methyl mercury toxicity were assessed in H. azteca. Biochemical endpoints that were evaluated include the inhibition of acetylcholinesterase enzyme and indicators of oxidative stress such as glutathione-S-transferase activity, lipid peroxidation, protein oxidation, and glutathione content. Methyl mercury antagonized the effects of chlorpyrifos in vivo on acetylcholinesterase inhibition. Methyl mercury did not induce oxidative damage; however, chlorpyrifos decreased glutathione-S-transferase activity. Additional studies demonstrated that methyl mercury did not affect the in vitro bioactivation of chlorpyrifos or the subsequent inhibition of acetylcholinesterase enzyme activity. Chemical-chemical interactions were examined utilizing chromatographic techniques. Results of thin layer chromatography suggested the formation of a chlorpyrifos-methyl mercury complex. The formation of this complex may result in increased accumulation of methyl mercury, apparent additive toxicity, and protection against chlorpyrifos mediated acetylcholinesterase inhibition.

Toxicological evaluation of constructed wetland habitat sediments utilizing Hyalella azteca 10-day sediment toxicity test and bacterial bioluminescence.

A toxicological evaluation was conducted on wetland habitats created as a result of run-off from agricultural areas. These temporary wetlands were created by using drop pipes as a means of reducing erosional cutting in agricultural fields. Toxicity bioassays utilizing bacterial bioluminescence and Hyalella azteca were used to assess sediment pore water and whole sediment, respectively. Inhibition of bacterial bioluminescence was initially used to determine relative toxicities of pore water from ten wetland sites. Constructed wetland sites were compared to the University of Mississippi Biological Field Station, a relatively pristine reference site. The H. azteca ten day sediment toxicity test was utilized to assess sediment from four selected sites using survival and growth as toxicological endpoints. Results from the toxicological evaluation, along with extensive ecological evaluations, were used to assess the best approach for implementation of temporary wetland habitats with existing agricultural practices.

Comparison of three histological fixatives on the immunoreactivity of mammalian P-glycoprotein antibodies in the sheepshead minnow, Cyprinodon variegatus.

Mammalian P-glycoprotein is a highly conserved integral membrane protein functioning as an energy-dependent efflux pump which decreases the concentration of certain lipophilic aromatic compounds entering the cell by diffusion. Expression of such a transporter in teleost species could play a significant role in conferring resistance to fish populations exposed to xenobiotic stressors and may serve as a potential indicator of species at risk for certain environmental contaminants. In previous studies we demonstrated that a strong correlation existed between corresponding mammalian and teleost tissues showing immunoreactivity to specific mammalian P-glycoprotein antibodies. In the present study, comparisons of staining pattern, intensity, and tissue specificity between tissues treated in Bouin's, Dietrich's and Lillie's histological fixatives were determined in the sheepshead minnow, Cyprinodon variegatus, using monoclonal antibodies C219, C494, JSB-1 and polyclonal antiserum MDR(Ab-1). Immunoreactivity of these antibodies was found to be fixative-dependent. Results are presented illustrating the differential staining patterns and tissue specificity observed for each tissue type, fixative, and antibody combination. Our data indicate tissue fixation has a significant impact on P-glycoprotein antibody immuno-reactivity in teleost tissues and must be considered in the comparison and interpretation of results.

Current diagnosis and treatment of corneal ulcers.

Successful treatment for a corneal ulcer requires proper diagnosis and antibiotic selection. The management should be guided by the severity of the clinical presentation, the ophthalmologist's confidence in making the proper diagnosis of bacterial keratitis, and the level of trust in the antibiotic agents chosen for the causative organisms. Universal standards include pretreatment cultures and dual broad-spectrum fortified antibiotics. Recent changes in practice suggest that empirical monotherapy treatment with a fluoroquinolone antibiotic may be appropriate for certain cases of bacterial keratitis. This article reviews the various diagnostic methods and treatment options currently practiced in the ophthalmic community.

Influence of beta-naphthoflavone and methoxychlor pretreatment on the biotransformation and estrogenic activity of methoxychlor in channel catfish (Ictalurus punctatus).

The organochlorine pesticide methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl) ethane] (MXC) has been classified as a proestrogen in mammals and fish, requiring demethylation prior to eliciting estrogenic activity or binding to the estrogen receptor. While microsomal demethylation occurs readily in the liver of fish, little is known about the enzyme(s) responsible or the effect of cytochrome P450 (CYP) inducers, other than those of CYP1A and CYP2K, on biotransformation. Consequently, male channel catfish were pretreated with MXC or beta-naphthoflavone (BNF), alone and in combination, to determine their effects on CYP protein expression, MXC biotransformation by hepatic microsomes, microsomal protein binding, and MXC estrogenic activity as determined by serum vitellogenin and 17beta-estradiol. Liver microsomes of both treated and untreated mature male catfish catalyzed formation of monodemethylated MXC, bisdemethylated MXC, as well as ring-hydroxylated metabolites. Pretreatment with BNF did not affect MXC metabolite profiles, overall rates of MXC biotransformation, or microsomal proteins recognized by anti-trout CYP2K1, but had the expected effect of inducing CYP1A and associated ethoxyresorufin O-deethylase activity. By contrast, pretreatment with MXC, alone or in combination with BNF, significantly reduced rates of MXC biotransformation and binding to liver microsomal protein. MXC/BNF cotreatment followed by MXC significantly induced serum vitellogenin, whereas MXC treatment alone led to a nonsignificant increase in vitellogenin and a significant increase in serum 17beta-estradiol. Thus, estrogenic activity elicited by cotreatment with MXC and BNF can occur despite diminished capacity of liver microsomes to catalyze formation of estrogenic demethylated metabolites or metabolites that bind microsomal protein. Possible mechanisms of MXC-induced attenuation of CYP-dependent metabolism are discussed.

Combined granular-lattice ('Avellino') corneal dystrophy.

BACKGROUND/PURPOSE: In 1988, a report was published describing the histopathologic examination of corneal buttons of 4 patients who had undergone unilateral keratoplasty because of decreased vision caused by what had been diagnosed clinically as granular dystrophy. But on pathologic examination, lesions characteristic of both granular dystrophy and lattice dystrophy were found in each of the 4 corneal buttons. The patients came from 3 different families, each of which traced its origin to the Italian province of Avellino. We studied the clinical and histopathological features of 4 corneas affected by combined granular-lattice dystrophy, adding thereby to the total of 12 other corneas that have been so-described in the literature. METHODS: Two women underwent bilateral penetrating keratoplasty for what was diagnosed clinically as Reis-Bücklers dystrophy in the first patient, and as granular dystrophy in the second patient. We studied all 4 corneas pathologically, using both conventional hematoxylin and eosin stains as well as special histochemical techniques. RESULTS: All 4 corneas contained lesions characteristic of both granular dystrophy and lattice dystrophy, a circumstance that has given rise to the name "combined granular-lattice dystrophy." The patients are not known to be of Italian ancestry. CONCLUSIONS: Three clinical signs characterize combined granular-lattice dystrophy: (1) anterior stromal discrete, grayish-white deposits; (2) lattice lesions located in mid-stroma to posterior stroma; and (3) anterior stromal haze. Both clinically and histopathologically, the lattice lesions are of greater diameter than are those that occur in lattice dystrophy type I. In the past few years, striking advances have been made in understanding the genetics of combined granular-lattice dystrophy. The most recent of these was published just 2 months before the 1997 meeting of the American Ophthalmological Society, and establishes a common molecular origin for granular dystrophy, lattice dystrophy type I, Avellino dystrophy, and Reis-Bücklers dystrophy.

Xenobiotic interaction with and alteration of channel catfish estrogen receptor.

In teleostean in vivo studies, the vitellogenin response to environmental estrogens is not completely predicted by mammalian literature. One possible explanation for differences is heterogeneity of the estrogen receptor (ER) structure between species. Therefore, ER from channel catfish (Ictalurus punctatus) hepatic tissue was characterized by binding affinity for several compounds. Affinity was indirectly measured as potency of the chemical for inhibiting binding of radiolabeled estradiol (E2) to specific binding sites. The order of potency among therapeutic chemicals was ethinylestradiol > unlabeled E2 = diethylstilbestrol > mestranol > tamoxifen >> testosterone. Unlabeled E2 had an IC50 of 2.2 nM. Several environmentally relevant chemicals were evaluated in a similar manner and the order of potency established was the o-demethylated metabolite of methoxychlor (MXC) > nonylphenol (NP) > chlordecone > MXC > o,p'-DDT > o,p'-DDE > beta-hexachlorocyclohexane. Demethylated MXC had an IC50 1000-fold greater than that of E2. Of the most potent inhibitors, NP appeared to be a competitive inhibitor for the same binding site as E2, while o-demethylated MXC had a more complex interaction with the receptor protein. ER from nonvitellogenic females was determined to have a Kd value of 1.0 to 1.3 nM. Because E2 has been reported to up-regulate teleostean ER, the hepatic ER population following in vivo xenobiotic exposure was assessed. NP significantly increased ER per milligram hepatic protein almost to the same extent as E2, but did not increase Kd to the same extent as E2.

Environmental estrogenic effects of alkylphenol ethoxylates.

Alkylphenol ethoxylates (APEs) and related compounds recently have been reported to be estrogenic because it has been demonstrated in laboratory studies that they mimic the effects of estradiol both in vitro and in vivo. Chemicals referred to as "environmental estrogens" are suspected of causing health effects in both humans and wildlife through disruption of the endocrine system. In this review, the occurrence, environmental fate, and biological effects of APEs are presented. To provide understanding of the potential for endocrine disruption due to environmental estrogens, the physiology of estrogens in mammals and fish is also reviewed. The estrogenic potency of other environmental estrogens is compared to the potency of APE degradation products. The reproductive effects of estrogenic compounds are considered when evaluating the potential health effects of APEs. Given the reported environmental concentrations and bioconcentration factors of APE products, the potential for these compounds to produce estrogenic effects in the environment appears low. Although questions concerning the physiological effects of APEs and other environmental estrogens remain unanswered, there are indications that research is in progress that will lead to better understanding of the risks to humans and wildlife.

Metals and school learning: a review of investigative techniques.

A growing body of literature suggests a relationship between metals and a threatened health status among children, adolescents, and adults. Recently, several investigators have reported a direct link between exposure to metal and diminished affective, behavioral, and cognitive performance of school children. The purpose of the present study was to review and analyze methods and procedures used to understand the relationship between exposure to metals and cognitive, social, and motor functioning of school children.

Relationship of aquatic natural organic material characteristics to the toxicity of selected insecticides.

Toxicities of the commercial insecticide formulations of azinphos methyl, chlorpyrifos, fenvalerate, and methyl parathion were evaluated in streamwater samples containing natural organic material (NOM) using a modification of a bacterial bioluminescence assay. Toxicity reduction of azinphos methyl was significantly (P < 0.05) correlated with water sample nonvolatile total solids (NVTS) concentration. Toxicity reductions of fenvalerate and of methyl parathion were significantly correlated to E4/E6 absorbance ratio of NOM, and to the proportion of NOM having molecular weight greater than 3700 Da. Toxicity reductions of fenvalerate and methyl parathion had significant correlations to the concentration of NVTS in water samples. Linear regressions of insecticide EC50 values to these NOM characteristics were used to mathematically model the effect of NOM on insecticide toxicity. Insecticide logKOW values were significantly correlated to the slopes of the EC50-total organic carbon (TOC) concentration regression lines. This confirms that the level of association of some nonionic organic contaminants with NOM is a function of the contaminant's water solubility. No single NOM characteristic was the best predictor of toxicity for all insecticides examined. However, these findings suggest that the influence of NOM on toxicity results primarily from differences in TOC content and abundance of larger molecular size NOM.

Detection of proliferating cell nuclear antigen in tissues of three small fish species.

An immunohistochemical assay for proliferating cell nuclear antigen (PCNA) identifies cells in all active phases of the cell cycle. In this study, PCNA methodology, which was developed primarily for mammalian tissues, was adapted to three small fish species, medaka (Oryzias latipes), guppy (Poecilia reticulata), and western mosquitofish (Gambusia affinis) that are used in carcinogenesis bioassays and environmental sentinel studies. Our study showed that PCNA can be identified in routinely processed, paraffin embedded specimens of these fishes. Optimum staining conditions were dependent on fixative, primary antibody, antigen retrieval processing, and protein blocking reagent. Best results were achieved using 10% neutral buffered formalin as the fixative, clone PC10 as the primary antibody, and a combination of powdered milk and bovine serum albumin as a protein block. Except for medaka specimens, antigen retrieval was not required for specimens preserved in 10% neutral buffered formalin, but was required for the other fixatives tested. In whole fish specimens, PCNA marked cells in normally proliferating tissues such as testis, ovary, primary filament epithelium of the gill, hematopoietic tissues, thymus, retina and alimentary tract. The study demonstrated the successful application of mammalian-based PCNA technology to these aquatic species. Further applications of the assay will aid in understanding the role of cell proliferation in normal, diseased, and toxicant-affected tissues of aquatic animals.