The development of an aquatic bivalve model: evaluating the toxic effects on gametogenesis following 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) exposure in the eastern oyster (Crassostrea virginica).

The objective of this study is to develop a gametogenesis protocol to serve as a model for evaluating the toxic effects of chemicals on oogenesis and spermatogenesis in the eastern oyster (Crassostrea virginica). The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) was selected as a "proof of principle" toxicant to examine developmental toxicity in this invertebrate system. The studies were designed to: (1) test the model using 2,3,7,8-TCDD and (2) to use histopathological evaluations to characterize the effects on oocyte and sperm development during stages of gametogenesis. 2,3,7,8-TCDD at 10 pg/g resulted in significant histopathological gonadal lesions by day 14 of gametogenesis in both female and male oysters. These lesions resulted in complete inhibition of gonadogenesis. Studies also showed that a total body dose of 2 and 10 pg/g 2,3,7,8-TCDD caused adverse responses resulting in abnormal gametogenesis in female and male oysters, respectively, such as: (1) incomplete oocyte division, (2) inhibition of oocyte growth and maturation, (3) unsynchronized sperm development, and (4) inhibition of spermatogenesis. The eastern oyster is one of the most responsive invertebrate models tested to date for reproductive effects of chemicals. Therefore, the eastern oyster can be used as a sensitive toxicological model for examining the effects of dioxin-like compounds and other xenobiotics on gametogenesis. The reported studies show that environmentally relevant concentrations of 2,3,7,8-TCDD (2-10 pg/g) have a significant adverse effect on oyster gametogenesis.

Hazard evaluation of diisononyl phthalate and diisodecyl phthalate in a Japanese medaka multigenerational assay.

Reproductive and developmental effects of diisononyl phthalate (DINP) and diisodecyl phthalate (DIDP) were evaluated in a Japanese medaka (Oryzias latipes) multigeneration protocol. Each phthalate was administered via fish flake diets at a concentration of 20 microg/g (1 microg/g fish/day). Two controls were included, untreated and acetone carrier. The F(0) and F(1) generation adults were reared to sexual maturation and the test was ended prior to sexual maturation of the F(2) generation. Biochemical, individual, and population parameters were evaluated: testosterone metabolism, 7-ethoxyresorufin-o-deethylase (EROD) activity, survival, development, growth, gonadal-somatic index, histopathology, sex ratio, and fecundity. Male fish showed a two-fold induction of several testosterone metabolites in the DINP-treated group compared to the untreated control but not the acetone control. In a similar manner, in female fish only the DIDP-treated group expressed greater testosterone hydroxylase activity. There were neither sex- nor treatment-related differences in the results from the EROD assay. A statistically significant transient delay in red blood cell pigmentation was observed. The male-to-female ratio was consistent across treatments and the phenotypic and histological gender classifications were in agreement. Egg production was not significantly different among treatment groups. Neither phthalate elicited an effect on reproduction or development at various levels of biological organization.

Effect of gestational and lactational 2,3,7, 8-tetrachlorodibenzo-p-dioxin exposure on the level and catalytic activities of hepatic microsomal CYP1A in prepubertal and adult rats.

We determined the inducibility, as well as the persistence of the induction, of hepatic microsomal CYP1A1 and CYP1A2 (by western blot analysis), and their catalytic activities (as measured by resorufin ether O-dealkylation) in prepubertal (25-day-old) and adult (120-day-old) offspring of timed-pregnant Sprague-Dawley rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD treatment was subcutaneous, at a low dose of 0.1 microg/kg, on gestational days 7, 14, and 20, and on lactational days 7 and 14. CYP1A1 protein was induced significantly (23-fold) in prepubertal but not in adult offspring of TCDD-exposed dams, whereas ethoxyresorufin O-deethylase (EROD) activity, which is CYP1A1-preferential, was induced less extensively (5-fold) and slightly (1.7-fold) in the prepubertal and adult offspring, respectively. Benzyloxyresorufin O-debenzylase (BROD) activity, which is CYP2B-preferential but has been reported to be catalyzed by CYP1A1, was also induced 5- and 6-fold in prepubertal and adult offspring, respectively, of TCDD-exposed dams. However, the induced BROD activity was neither inhibited by antibody against CYP1A1 nor accompanied by an elevated level of microsomal CYP2B. CYP1A2 was induced slightly only in prepubertal offspring of TCDD-treated dams. There was suggestive evidence of enhanced lipid peroxidation in hepatic microsomes from prepubertal but not adult offspring of TCDD-treated dams. These data showed that in utero plus lactational TCDD exposure effected transient induction of hepatic microsomal CYP1A1 but sustained induction of BROD activity, which may be catalyzed by enzymes other than CYP1A or CYP2B.

Chemical and physical properties of processed newspaper compared to wheat straw and wood shavings as animal bedding.

Because of continuing concerns about the safety and the suitability of recycled newspaper as an animal bedding material, municipal curbside-collected newspaper was processed into chopped and pelleted forms for comparison studies with wheat straw and kiln-dried pinewood shavings. Measurements included nutrient, heavy metal, dioxin and furan content, particle size distribution, density, combustion potential, and water-holding capacity. Recycled newspaper, straw, and wood shavings tested below or equivalent to National Research Council dietary tolerance levels and US Environmental Protection Agency toxic equivalent levels. Small particle size distribution was shavings > straw > all forms of newspaper. The density of pelleted newspaper was 50-fold greater than that of chopped newspaper and straw and 15-fold greater than shavings. In simulated flash burns, chopped newspaper, straw, and shavings ignited, and flames spread rapidly in newspaper and shavings and lasted the longest in shavings. Pelleted newspaper did not ignite. Chopped and pelleted forms of newspaper and wood shavings had higher water holding capacities (>400%) than did straw (200%). Animal industries can, in confidence, utilize recycled newspaper as an animal bedding material, providing that sources of low toxicity are identified, and suitable processed forms are produced.

A proposed multigeneration protocol for Japanese medaka (Oryzias latipes) to evaluate effects of endocrine disruptors.

Definitive data on reproductive impairment of chronically exposed populations may be required to assess the appropriateness of the existing test methods for hazard identification and prioritization of endocrine modulators. Multigeneration toxicity testing protocols for wildlife receptors are lacking. To help address this gap we describe a multigeneration fish assay using the freshwater fish, Japanese medake (Oryzias latipes). This test species has been used for the evaluation of carcinogenic, teratogenic and reproductive effects and is sensitive to estrogen exposure producing ovo-testis, altered biochemical parameters and phenotypic characteristics. Due to the short life cycle, a multigeneration test with medaka can be conducted in 1 year. Endpoints evaluated include: survival, growth, sex ratio, fecundity, embryonic lesion occurrence, embryonic stage development, gonadal and hepatic somatic indices, histopathology and biochemical parameters. As new endpoints are developed they can be incorporated into the protocol. Results of a positive control (17 beta-estradiol) study are presented to give an indication of the baseline associated with various test endpoints and to highlight the importance of nutrition in the experimental design. 17 beta-Estradiol treatment induced vitellogenin production in male and female medaka, feminized males, and disrupted egg production. The proposed protocol provides researchers with an effective multigeneration fish test that can be used to examine potential effects of stressors at the population, individual, cellular and subcellular level.

Using capillary electrophoresis to study the chemical conditions within cracks in aluminum alloys.

The environment-assisted cracking (EAC) susceptibility of some aluminum alloys used for airplane structural components currently limits their use in the peak strength condition. Understanding the mechanism of EAC will facilitate the development of crack-resistant alloys with optimum mechanical properties. One component towards understanding the fundamental processes responsible for EAC is a comprehensive knowledge of the chemical conditions within cracks. The present work uses capillary electrophoresis (CE) to quantify the crack chemistry in order to provide insight into the nature of the mechanism controlling cracking. The highly restricted geometry of cracks in metals means that a crack typically contains less than 10 microliters of solution. The high mass sensitivity combined with the inherently robust nature of CE makes it an ideal analytical technique for this application. Complicating factors in the accurate determination of the crack environment include high levels of sodium present from the test solution. Low sample volume and analyte matrix complexity necessitated the development of specific sampling, extraction and analysis methods. Analysis of the crack solutions in EAC-susceptible material revealed high levels of Al3+, Mg2+, Zn2+, and Cl- near the crack tip. Cations arise from the anodic dissolution of the alloy, whereas chloride ingress from the external environment occurs to maintain solution electroneutrality within the crack. In contrast, EAC-resistant material exhibited significantly lower concentrations of dissolution products.

Effects of polychlorinated biphenyls on thyroid hormones and liver type I monodeiodinase in the chick embryo.

Polychlorinated biphenyls (PCBs) are widespread environmental contaminants which can biomagnify to higher tropic level organisms including birds. Circulating thyroid hormones (TH) and growth are decreased by PCB exposure. The first set of studies investigated the effects of PCBs on an enzyme responsible for TH homeostasis, hepatic type I monodeiodinase (MDI) in chicken embryos. Fertile chicken eggs were injected with Aroclor 1242, Aroclor 1254, 2,2',6, 6'-tetrachlorobiphenyl (TCB), 3,3',4,4'-TCB, or 3,3',5,5'-TCB on Day 0 and studies were terminated on Incubation Day 21. Hepatic MDI activity was reduced in embryos treated with the Aroclor mixtures. No effects on MDI activities were observed after PCB isomer treatment. Liver weights from embryos treated with Aroclor 1242 were decreased. In the second study, chick embryos were exposed to these same PCBs in order to evaluate their effect on circulating THs and growth. Treatment with PCBs had no effect on body weight. Femur length were decreased with Arcolor 1242 treatment. A decrease in plasma concentration of thyroxine was observed after treatment with Aroclor 1242 and Aroclor 1254. Based on these findings, it is evident that PCBs alter the thyroid axis. Bird circulating TH levels, which are generally reported, may not be a good biomarker for low-dose exposure to PCBs. However, the reduction in MDI activity was more sensitive to PCB mixture exposure and may be a useful biomarker.

PCDD and PCDF contamination in catfish feed from Arkansas, USA.

One combined catfish feed sample from Arkansas, USA, and its eight ingredients were analyzed for PCDDs and PCDFs. One of the ingredients, soybean meal, was highly contaminated by PCDDs, especially the toxic 2,3,7,8-substituted congeners, e.g., 7.3 pg/g dry weight or 370 pg/g lipid for the 2,3,7,8-tetra CDD. The I-TEQ value for the soybean meal was 11.4 pg/g dry weight or 576 pg/g fat. The corresponding values for the combined catfish feed concentrations were approximately 3 times lower. The congener pattern, the congener profile and the ratio sigma PCDDs/sigma PCDFs for the soybean meal were quite unique. We are not aware of any environmental sample or technical product with similar characteristics. As a result, natural formation of the PCDDs found in the soybean meal cannot be ruled out.

Effects of polychlorinated biphenyl mixtures and three specific congeners on growth and circulating growth-related hormones.

Polychlorinated biphenyls (PCB) are ubiquitous environmental contaminants that bioaccumulate in avian species. Exposure to PCBs can result in decreased growth. Thyroid hormones and growth hormone (GH) are important for normal growth. The present studies employed the chicken embryo to investigate effects of Aroclor 1242, Aroclor 1254, 2,2',6,6'-tetrachlorobiphenyl (TCB), 3,3',4,4'-TCB, and 3,3',5,5'-TCB on growth and growth-related hormones. The following indices were measured: embryo mortality, body weights, bone length, pituitary GH content, and plasma concentrations of triiodothyronine (T3), thyroxine (T4), GH, and insulin-like growth factor-1 (IGF-1). Fertile eggs were injected with PCBs on Day 0 and indices determined on Day 17 of incubation. Unexpectedly, 3,3',5,5'-TCB or low-dose Aroclor 1242 treatment increased body weight and bone length (P < 0.05), whereas Aroclor 1242 (high dose), 3,3,4,4'-TCB, or Aroclor 1254 treatment reduced body weights and/or bone length (P < 0.05). Aroclor 1242 or 3,3',4,4'-TCB (low-dose treatment) elevated plasma T4 concentrations (P < 0.05). Both growth and pituitary GH content were increased (P < 0.05) by 3,3',5,5'-TCB (low dose) or Aroclor 1242 treatment. Despite marked differences in growth rates, plasma T3, GH, and IGF-I concentrations were unaffected by PCB treatment. Growth-related hormones may not be responsible for the growth depression observed after PCB treatment. Possibly the decrease in growth occurred because of general toxicity. The importance of chlorine position in causing thyroid hormone axis alterations was not clearly established.

Polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/PCDF) in food samples collected in southern Mississippi, USA.

In 1994, we analyzed 43 foodstuff samples from local supermarkets in southern Mississippi, USA, for PCDD/PCDF. 2,3,7,8-Cl4DD could be quantified in 31 of these samples. On a lipid basis, levels in meat (0.53-1.10 pg I-TEQ/g) and dairy products (0.42-1.10 pg I-TEQ/g) were slightly lower than those reported from other industrialized countries. While levels in dairy samples from the United States and Europe are comparable, there is a difference in the contribution of individual congeners to the I-TEQ: for example, in milk samples from Germany approximately 40% of the I-TEQ is due to the presence of 2,3, 4,7,8-Cl5DF while in the Mississippi samples this congener only contributes 16%. The highest concentrations of PCDD/PCDF in our study were detected in the farm-raised catfish (10.2-27.8 pg I-TEQ/g). A unique finding was that in addition to the 2,3,7,8-substituted PCDD/PCDF the catfish samples contained many non-2,3,7,8-substituted congeners. This is unusual because vertebrate animals selectively eliminate or metabolize the non-2,3,7,8-substituted congeners.

The effect of levenogesterol (Norplant) contraception on coagulation as measured by antithrombin-III levels.

PURPOSE: To determine the effect of contraception given immediately postpartum on coagulation as measured by antithrombin III. STUDY DESIGN: In this prospective study, parturients (n = 85) self selected three means of postpartum contraception: levenorgesterol implants, oral contraceptives, or a barrier method. RESULTS: Baseline coagulation was assessed by antithrombin-III levels in each of the 85 women within 48 hours of delivery (100.35 +/- 1.61%) and at one (109.1 +/- 1.89%) and six (105.51 +/- 1.71%) weeks postpartum. There was a rise in antithrombin-III after delivery but there were no significant differences between the groups. CONCLUSION: The levenorgesterol implant system did not cause a decrease in antithrombin-III in normal parturients.

PIP: The effect of postpartum Norplant implant use on coagulation factors was investigated in a prospective study conducted in Mississippi, US. 85 postpartum women were given a choice of 3 contraceptive methods: levonorgestrel implants (n = 25), oral contraceptives (n = 38), or a barrier method (n = 22). Antithrombin-III (AT-III) levels were measured on the day of discharge from the hospital after delivery, 7 days after delivery, and 6 weeks after delivery. There was no significant difference between groups in the initial AT-III level (mean, 100.35 +or- 1.61%). At the end of the first postpartum week, AT-III levels rose significantly to 109.1 +or- 1.89%, but again, there were no significant differences according to contraceptive method. By the 6-week follow-up, AT-III levels had stabilized at a mean of 105.51 +or- 1.71%, with no significant between-group differences. AT-III levels in pill users declined more markedly between the first and sixth postpartum weeks than those in the 2 other groups. These findings indicate that subdermal implant use does not affect coagulation, and these devices can be inserted safely during the postpartum period.

Alternative models for low dose-response analysis of biochemical and immunological endpoints for tetrachlorodibenzo-p-dioxin.

As part of the current reassessment of dioxin, the empirical relationships between administered tetrachlorodibenzo-p-dioxin and selected immune and biochemical endpoints were investigated. A dose-response analysis from the published literature was performed using Linear, Sigmoid-Emax and Power Law functions. The results of this analysis indicate that the use of a wide dose range may bias the interpretation of low-dose phenomenon. The Power Law function was applied exclusively to low-dose subsets enabling estimation of dose response in the low-dose range. Subsequently, high-dose data were fit using Power Law subset analysis. This approach resulted in a change in slope value from low- to high-dose subsets for thymic atrophy, immune suppression, benzo(a)pyrene hydroxylase activity, and ethoxyresorufin-o-deethylase activity. This change suggests that there is a high probability that there is a tissue concentration along the dose-response continuum which results in biological activity from low to high dose. This analysis also demonstrates that the Power Law functional fit to the low-dose data differs quantitatively from the fit to the high-dose data for several noncancer endpoints. Therefore, by using the Power Law function a more accurate and biologically relevant assessment of risk can be produced for non-cancer endpoints.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on benzo(a)pyrene hydroxylase activity in embryos of the Japanese medaka (Oryzias latipes).

When Japanese medaka embryos were exposed to 12 ng/l 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) beginning on the day of fertilization (day 0), benzo(a)pyrene hydroxylase (B(a)PH) activity was induced in the whole embryo 105,000 g fraction by day 5 of development, which coincided with liver development. The induction of B(a)PH activity also coincided with the appearance of 2,3,7,8-TCDD induced hemorrhagic and edematous lesions. B(A)PH induction only occurred in embryos exposed to toxic concentrations (greater than 10 ng/l) of 2,3,7,8-TCDD. B(a)PH induction also occurred in embryos after exposure to 10 ng/l 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 50 micrograms/l 1,2,7,8-TCDD. Both 2,3,7,8-TCDF and 1,2,7,8-TCDD are toxic to Japanese medaka embryos at concentrations that resulted in the induction of B(a)PH activity. B(a)PH activity was not induced by the non-toxic congener 1,3,6,8-TCDD at concentrations as high as 50 micrograms/l. The structure activity relationship for B(a)PH induction in Japanese medaka embryos was similar to that which is observed in other species and biological systems, suggesting that the biological activities of these compounds may also be mediated through the putative Ah receptor in these fish embryos. At 50 micrograms/l, beta-naphthoflavone (BNF) induced B(a)PH activity in Japanese medaka embryos to similar levels as 2,3,7,8-TCDD did at toxic concentrations. However, at 50 micrograms/l, BNF was not toxic to Japanese medaka embryos. Therefore, the induction of B(a)PH activity probably did not directly result in the toxicity observed in these fish embryos after exposure to 2,3,7,8-TCDD.

Ibuprofen prevents deterioration in static transpulmonary compliance and transalveolar protein flux in septic porcine acute lung injury.

UNLABELLED: The effects of intravenous ibuprofen on measurements of pulmonary function and alveolar capillary membrane permeability to protein in sepsis-induced porcine acute lung injury (ALI) were studied. Young swine (15-25 kg) were anesthetized, cannulated, and ventilated (5 cm H2O PEEP, 0.5 FIO2, and 15 cc/kg tidal volume). Three groups were studied: septic animals (Ps, n = 10) received Pseudomonas aeruginosa for 1 hr IV, controls (C, n = 9) received 0.9% NaCl, and ibuprofen-treated septic animals (Ps + Ibu, n = 7) received ibuprofen 12.5 mg/kg at 0 and 120 min post Ps. Systemic (SAP) and pulmonary (PAP) arterial pressures, PaO2, cardiac index (CI), static lung compliance (CL), EVLW (thermal cardiogreen), and peripheral white blood cell counts (WBC) were measured. Bronchoalveolar lavage (BAL) was performed for protein and % neutrophil (%PMN) content. RESULTS: Ps produced significant (p less than 0.05) decreases in CL, PaO2, SAP, CI, and peripheral WBC and increases in PAP, EVLW, BAL protein, and %PMN's vs. controls. Ibu prevented the early increase in PAP and attenuated the late increase in PAP and EVLW. Ibu also maintained PaO2, CL, BAL protein, and %PMN's in BAL at control levels, but exhibited no significant effect on peripheral leukopenia. These data strongly suggest that ibuprofen administered before and at 120 min after onset of Pseudomonas infusion improves lung compliance and affects neutrophil function sufficiently to significantly ameliorate many of the physiologic derangements in acute sepsis.

Pulmonary compliance: early assessment of evolving lung injury after onset of sepsis.

We compared the sensitivity of dynamic (Cdyn) and static lung compliance (CL) with indicators of permeability injury in a model of septic porcine adult respiratory distress syndrome. Two groups of anesthetized ventilated swine (15-25 kg) were studied. Septic animals (Ps, n = 13) received Pseudomonas aeruginosa intravenously for 1 h, which resulted in severe adult respiratory distress syndrome. Controls (C, n = 13) received 0.9% NaCl. Cdyn, CL, bronchoalveolar lavage for protein estimation, and thermal cardiogreen extravascular lung water (EVLW) measurements were performed in seven C and eight Ps animals. Six C and five Ps animals underwent gamma camera measurement of lung-to-heart ratio (slope index) of 99Tc-labeled human serum albumin. Both Cdyn and CL decreased significantly (P less than 0.01) at 30 min and thereafter in Ps vs. C. EVLW, slope index, and bronchoalveolar protein content increased significantly (P less than 0.05) in Ps vs. C at 120, 150, and 300 min, respectively. Cdyn and CL decreased well before onset of permeability injury. These early changes may be due to release of vasoactive mediators and sequestration of neutrophils in the pulmonary capillaries and later to increases in EVLW. Measurement of Cdyn and CL represents an early means of assessing evolving lung injury in this acute septic porcine model.

Interactive inhibition of erythroid 59Fe utilization by benzene metabolites in female mice.

Using radioiron uptake into erythrocytes as a measure of hematopoiesis, it was demonstrated that benzene inhibited bone marrow function in female mice. Hydroquinone was marginally effective, but the inhibition occurred only at the highest dose tested (100 mg/kg). The combination of phenol and hydroquinone was more effective in reducing erythrocyte production than either chemical given alone. Catechol given alone was not inhibitory but when phenol was added to catechol, erythropoiesis was suppressed, as observed for the phenol and hydroquinone combination. It appears that benzene toxicity may be the result of cooperative inhibitory effects produced by its metabolites.

Benefit of nasal CPAP in obstructive sleep apnea is due to positive pharyngeal pressure.

The purpose of this study was to determine if the mechanism of nasal continuous positive airway pressure's (CPAP's) effectiveness is to act as a pneumatic splint or to increase functional residual capacity (FRC) and consequently, upper airway caliber. Four subjects with obstructive sleep apnea underwent 3 nights of polysomnography: night 1, control; night 2, nasal CPAP; night 3, external subatmospheric pressure (ESAP). ESAP, a negative pressure body suit, increases FRC. We measured the changes in FRC with nasal CPAP and ESAP using the weighted spirometer technique. The dose used for the ESAP night was the dose that produced the same FRC as the subject's prescribed nasal CPAP dose. The mean number of arousals and the respiratory events index were higher on ESAP and control nights. Less severe oxygen desaturation occurred during non-rapid-eye-movement sleep on the nasal CPAP and ESAP nights. These preliminary results show that increasing FRC alone does not account for the effectiveness of nasal CPAP, and splinting of the collapsible upper airway is necessary.