Alterations in immune function and CYP450 activity in adult male deer mice (Peromyscus maniculatus) following exposure to benzo[a]pyrene, pyrene, or chrysene.

Polycyclic aromatic hydrocarbons (PAHs) are among the most common classes of chemical contaminants found at hazardous waste sites. Deer mice (Peromyscus maniculatus) exhibit a wide geographic distribution throughout North America and have been suggested as a terrestrial biomonitoring species to facilitate comparisons between superfund sites. Chemicals tested were benzo[a]pyrene (BaP; CAS number 50-32-8), pyrene (Pyr; CAS number 129-00-0), and chrysene (Chr; CAS number 218-01-9). Adult male deer mice were exposed via intraperitoneal (i.p.) injection every other day for 11 d to the PAHs (0.3, 1, 3, 10, or 30 mg/kg) or a corn oil carrier control. Both BaP and Chr suppressed the plaque-forming cell (PFC) response at all treatment levels. Pyr exposure (1-30 mg/kg) also resulted in suppression of this response. Macrophage pinocytosis was suppressed only by Chr (3, 10, and 30 mg/kg). Concanavalin A-induced proliferation was stimulated by BaP at all dose levels, by Pyr at 1-30 mg/kg, and by Chr at 30 mg/kg. Chr did not affect pokeweed mitogen (PWM)-induced proliferation; however, BaP (1-30 mg/kg) and Pyr (0.3-30 mg/kg) produced stimulation of this response as compared to respective controls. BaP and Chr stimulated cytochrome P-450 1A1 (CYP1A1) activity (3, 10, or 30 mg/kg) as measured by ethoxyresorufin O-deethylase (EROD) activity, but Pyr did not. These results indicate that immune function endpoints appear to be more sensitive to these PAHs than measured hepatic CYP450 activity.

Effects of binary mixtures of six xenobiotics on hormone concentrations and morphometric endpoints of northern bobwhite quail (Colinus virginianus).

This study investigated the effects of six endocrine disrupters in five different doses (0.1, 0.3, 1, 3, 10 mg/kg or microg/kg) in ethanol administered by oral gavage to bobwhite quail eggs. Six eggs each were in each dose group of coumestrol, ethynyl estradiol, indole-3-carbinol, o,p'-DDE, p,p'-DDE, or TCDD. Eggs were also dosed in two sets. One set was ethynyl estradiol (0, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0 microg/kg) and TCDD (0, 0.003, 0.01, 0.03, 0.1, 0.3 microg/kg). This set was dosed below the air cell with corn oil as vehicle. Also, northern bobwhite quail eggs were injected in ovo with nine binary mixtures of six xenobiotics prior to incubation (coumestrol (0.3 mg/kg), ethynyl estradiol (3.0 microg/kg), indole-3-carbinol (3.0 mg/kg), o,p'-DDE (1.0 mg/kg), p,p'-DDE (1.0 mg/kg), TCDD (0.1 microg/kg)). The mixtures injected were p,p'-DDE+indole-3-carbinol, coumestrol+indole-3-carbinol, TCDD+indole-3-carbinol, p,p'-DDE+o,p'-DDE, p,p'-DDE+ethynyl estradiol, coumestrol+ethynyl estradiol, coumestrol+TCDD, o,p'-DDE+ethynyl estradiol, TCDD+ethynyl estradiol. Eggs were dosed once prior to initiating incubation. Quail were allowed to hatch and were sacrificed at 21 days of age. Blood, measurements, and tissues were collected. Survival was significantly affected by increasing concentrations of TCDD in ethanol as revealed by trend analysis. Survival was also affected significantly by o,p'-DDE in ethanol but not by trend. Survival results of mixtures indicate significant differences among mixture, mixture components, and controls for coumestrol+TCDD, ethynyl estradiol+TCDD, and indole-3-carbinol+TCDD. Some trends from doses of single compounds that are supported by results in the literature were observed for hatchling weight of ethynyl estradiol dosed females, weight gain of indole-3-carbinol dosed males, weight gain and liver somatic index of o,p'-DDE dosed males, spleen somatic index of TCDD dosed males, and weight gain, gonad somatic index and egg gland somatic index of TCDD dosed females. In conclusion, the dose response treatments appeared to have effects beyond effects on survival of in ovo dosed quail. For mixtures, plasma estradiol concentrations were significantly different among coumestrol+ethynyl estradiol, ethynyl estradiol, coumestrol, and vehicle treatments. Liver somatic index among the same treatments was also significantly different. Kidney somatic index among ethynyl estradiol+p,p'-DDE, ethynyl estradiol, p,p'-DDE, and vehicle treatments was significantly different. Plasma estradiol and plasma testosterone ratios were very different among o,p'-DDE+p,p'-DDE, o,p'-DDE, p,p'-DDE, and vehicle treatments. Coumestrol and ethynyl estradiol appear antagonistic for plasma estradiol concentrations and liver somatic index when both chemicals are present together. Ethynyl estradiol and p,p'-DDE appear to act additively on kidney somatic index when combined together. Mixtures of compounds, used in this study indicate effects very different from either or both mixture components, indicating the lack of predictability of chemicals when combined in mixtures.

Identification of cytochrome P450 1B-like sequences in two teleost fish species (scup, Stenotomus chrysops and plaice, Pleuronectes platessa) and in a cetacean (striped dolphin, Stenella coeruleoalba).

The cytochromes P450 (CYP) constitute a multigene family of enzymes playing a critical role in the oxidation of many endogenous and xenobiotic substrates. The CYP1 family is of particular interest in environmental toxicology because its members are dominant in the metabolism of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and aryl amines. Three members of the CYP1 family, CYP1A1, CYP1A2, and CYP1B1, have been identified in mammals. We report here on the identification and cloning of cytochrome P4501B-like sequences from two teleost fish species and a marine mammal. Sequences clustering with CYP1B1 in phylogenetic analysis were obtained from liver cDNA of scup (Stenotomus chrysops), genomic DNA of plaice (Pleuronectes platessa), and liver cDNA of striped dolphin (Stenella coeruleoalba).

Modulation of endocrine pathways by 4,4'-DDE in the deer mouse Peromyscus maniculatus.

4,4'-DDT and 4,4'-DDE are widespread environmental contaminants that cause eggshell thinning in birds, altered sex ratios in the American alligator, and changes in the anal-genital distance in rodents. These contaminants are known to cause some of their toxicity by altering steroid receptor-mediated mechanisms. However, chemical-specific alterations in the expression of hormone-metabolizing enzymes may also be a mechanism for endocrine disruption, by altering the half-life of hormones in critical tissues. Previously, we showed that 4,4'-DDE causes a dose-dependent increase in ethoxyresorufin-O-deethylase (EROD) activity, but not pentoxyresorufin-O-dealkylase (PROD) activity, in the deer mouse. In this study, we demonstrated that 4,4'-DDE elicited a corresponding increase in CYP1A protein expression but not CYP2B using Western blotting and immunoprecipitation. 4,4'-DDE-mediated changes in phase II conjugating enzymes; UDP-glucuronosyltransferase (UGT) and phenolsulfotransferase (ST), were also investigated for the first time. Prepubescent female deer mice were dosed with 4,4'-DDE by gavage on days 1 and 2, then euthanized on day 4. As anticipated, dose-dependent increases in hepatic EROD and MROD activities, but not PROD or BROD, were observed. UGT activity was monitored by incubating liver microsomes and 14C-UDP-GA with potential substrates and measuring incorporation of radioactivity into TLC-resolved glucuronides. Dose-dependent increases in conjugation were observed with p-nitrophenol (a general UGT substrate) but not testosterone. Interestingly, a biphasic dose-response curve was observed for ST activity, with a peak at the 3 mg/kg dose. Dose-dependent increases in CYP1A1 and UGT-specific immunoreactive proteins were observed, suggesting de novo synthesis as a consequence of 4,4'-DDE exposure. We also measured Phase I and II enzymes in deer mouse platelets. Preliminary results indicate that the 4,4'-DDE-induced changes in liver Phase I and II enzyme activity were similar, but not identical, to those found in platelets. These results indicate that environmentally-relevant levels of 4,4'-DDE modulate the activity and expression of CYP1A1 and phase II enzymes in the deer mouse and that certain changes may be measured non-lethally.

Relative distribution of polychlorinated biphenyls among tissues of neonatal American alligators (Alligator mississippiensis).

Recent papers have investigated the utility of chemical analysis of the chorioallantoic membrane (CAM) as a nonlethal indicator of avian and reptilian exposure to persistent environmental contaminants. This study was undertaken to evaluate the chemical distribution among fat, CAM, and residual yolk tissues of live neonatal American alligators (Alligator mississippiensis) to investigate the potential utility of CAM use as a nonlethal indicator of exposure to polychlorinated biphenyls (PCBs). CAMs were collected from neonatal alligators at hatch, whereas yolk sacs and fat tissues were taken from each animal at euthanization (3 weeks posthatch). All collected tissue samples were separately Soxhlet extracted and analyzed by GC/ECD for PCBs. Log normalized, individual PCB congener concentrations in CAMs were significantly correlated with concentrations in fat (r(2) = 0.62) and yolk (r(2) = 0.56) tissues. Coefficients of determination from comparisons of homologue group concentrations varied from 0.13 to 0.90. Residue levels in neonatal oviparous organism tissues may be qualitatively assessed through chemical analyses of CAM tissues removed from discarded eggshells.

Effects of in ovo exposure to 2,3,7,8-TCDD on F1 generation adult chickens (Gallus gallus).

White Leghorn chickens (Gallus gallus) were used as surrogate species for the resident wild turkeys found on the Times Beach, Missouri, Superfund site. Parental chickens were injected with concentrations of 2,3,7,8-TCDD which modeled soil concentrations before (200 ppb) and after remediation (1ppb)[1]. Offspring were followed through development to assess alterations in reproductive maturity through the use of a four-way breeding study. F1 adult females exposed to a maternal dose of 8.6 ng/day began egg production approximately two weeks later than did F1 control adult females. By week eight, however, egg production between groups was equivalent. No differences were observed in eggshell gland estrogen or progesterone receptor levels.

Influence of roughage and grain processing in high-concentrate diets on the performance of long-fed Holstein steers.

The effects of roughage source and timing of inclusion in high-concentrate diets on feedlot performance were evaluated in each of 2 yr with 144 and 120 long-fed Holstein steers, respectively. Roughage, when included in the diet, was delivered in a built-in-roughage (BIR) pellet supplying 15 and 6% roughage (percentage of DM) or hay crop silage (HCS) supplying 7 and 10% roughage (percentage of DM) for the growing and finishing periods, respectively. The six dietary treatments (two pens each) investigated were continuous whole corn and pelleted supplement (no added roughage; WSC-PEL); BIR with cracked corn either in the growing (BIR-G) or finishing (BIR-F) period with whole corn being fed with pelleted supplement when BIR was not part of the diet; BIR and cracked corn fed continuously (BIR-CONT); and two HCS treatments in which the corn was fed either whole (HCS-WSC) or cracked (HCS-CSC) for the entire feeding period. Replicates were slaughtered 4 and 3 wk apart for yr 1 and 2, respectively. Pooled daily gains (yr 1 and 2) for the entire trial (1.39 to 1.45 kg/d) were not different (P > .05). Daily DM and concentrate intakes were lower (P < .001) and gain efficiency was higher (P < .001) for the WSC-PEL than for the other treatments. Carcass characteristics were similar among treatments except for an improved yield grade (P < .02) for the HCS treatments over the BIR treatments. The WSC-PEL treatment was the most profitable (+/steer) and the BIR-CONT diet was the least profitable. The HCS treatments were the most profitable of the roughage treatments. A newly proposed intake prediction equation for feedlot cattle performed similarly to the 1984 NRC equation with long-fed Holstein steer calves.

Toxicological foundations of ecological risk assessment: biomarker development and interpretation based on laboratory and wildlife species.

Ecological risk assessments based on chemical residue analysis and species demographics tend to ignore the bioavailability and bioaccumulation of the chemicals of concern. This study describes the incorporation of mechanistically based biomarkers into an ecological risk assessment of a poly-cyclic aromatic hydrocarbon (PAH)-contaminated site. A combination of soil residue analysis, tissue residue analysis, biomarkers in one-site trapped animals and biomarkers in animals confined to enclosures was used. In particular, the use of captured deer mice (Peromyscus maniculatus) for these studies is compared to the use of laboratory-raised deer mice placed in enclosures. This study indicates that the higher degree of variability in the responses of wild deer mice make the use of enclosure studies advantageous. Positive control studies performed by dosing laboratory-raised deer mice with the same PAHs as found on the site were used to validate this approach. These studies indicate that immune suppression occurred at PAH concentrations an order of magnitude below those required for the induction of ethoxyresorufin-O-dealkylase activity.