Reverse line blot probe design and polymerase chain reaction optimization for bloodmeal analysis of ticks from the eastern United States.

Determining the host preference of vector ticks is vital to elucidating the eco-epidemiology of the diseases they spread. Detachment of ticks from captured hosts can provide evidence of feeding on those host species, but only for those species that are feasible to capture. Recently developed, highly sensitive molecular assays show great promise in allowing host selection to be determined from minute traces of host DNA that persist in recently molted ticks. Using methods developed in Europe as a starting-point, we designed 12S rDNA mitochondrial gene probes suitable for use in a reverse line blot (RLB) assay of ticks feeding on common host species in the eastern United States. This is the first study to use the 12S mitochondrial gene in a RLB bloodmeal assay in North America. The assay combines conventional PCR with a biotin-labeled primer and reverse line blots that can be stripped and rehybridized up to 20 times, making the method less expensive and more straightforward to interpret than previous methods of tick bloodmeal identification. Probes were designed that target the species, genus, genus group, family, order, or class of eight reptile, 13 birds, and 32 mammal hosts. After optimization, the RLB assay correctly identified the current hostspecies for 99% of ticks [Amblyomma americanum (L.) and eight other ixodid tick species] collected directly from known hosts. The method identified previous-host DNA for approximately half of all questing ticks assayed. Multiple bloodmeal determinations were obtained in some instances from feeding and questing ticks; this pattern is consistent with previous RLB studies but requires further investigation. Development of this probe library, suitable for eastern U.S. ecosystems, opens new avenues for eco-epidemiological investigations of this region's tick-host systems.

Susceptibility to phenobarbital promotion of hepatotumorigenesis: correlation with differential expression and induction of hepatic drug metabolizing enzymes in heavy and light male (C3H x VY) F1 hybrid mice.

Higher body and carcass (body - liver) weights in sodium phenobarbital (PB) treated mice correlate with formation of multiple hepatocellular adenomas in yellow Avy/A and agouti A/a (C3H x VY) F1 hybrid male mice. To assess differences in PB induction of hepatic drug metabolizing enzymes, yellow Avy/A (C3H x VY) F1 hybrid male mice were fed 0.05% sodium PB in NIH-31 diet for 7 months. Livers from the heaviest and lightest mice in the untreated and PB groups were assayed. Total cytochrome P450 content, cytochrome P450IA-selective 7-ethoxyresorufin-O-deethylase and P450IIIA-selective testosterone-6 beta-hydroxylase activities were preferentially induced in the light mice. In contrast, P450IIB-selective 7-pentoxyresorufin-O-dealkylase activity was increased only 3-fold by PB in the light mice but 6-fold in the heavy mice. Testosterone UDP-glucuronyltransferase and gamma-glutamyltranspeptidase activities were induced in the light mice but not in the heavy mice. Glutathione-S-transferase N1:1-dependent activity was induced preferentially in the heavy mice. Significant differences also occurred in constitutive expression of P450IIIA-selective testosterone-6 beta-hydroxylase, P450IA-selective 7-ethoxyresorufin-O-deethylase and testosterone UDP-glucuronyltransferase activities between the untreated weight groups. Thus, expression of constitutive and PB-inducible forms of hepatic drug metabolizing enzymes differs between heavy and light Avy/A (C3H x VY) F1 hybrid subpopulations. This suggests that differential susceptibility to PB promotion of hepatocellular adenomas among genetically identical mice is accompanied by differences in the regulation of gene expression.

Postnatal function following prenatal reserpine exposure in rats: neurobehavioral toxicity.

Behavioral and neurochemical analyses were conducted on preweanling CD rats prenatally exposed to either 0, 0.375 or 0.750 mg/kg/day reserpine SC on gestation days 12-15. Offspring body weights were taken on test days, and pups were tested for negative geotaxis responding on postnatal day 8, developmental activity on days 12, 16 and 20, and auditory startle habituation on day 19 or 20. In addition, brains were removed from culled pups on day 1, 1 male and 1 female/litter on day 8, and animals tested for activity on day 21. Neurochemical assays were performed on whole brains from 1- and 8-day-old pups, and on caudate nucleus, frontal cortex and hippocampus of day 21 rats. Treatment resulted in dose-related decreases in maternal weight gain over gestation and mean pup weight at birth. Changes in the normal developmental activity pattern were both sex and dose dependent in treated rats. In auditory startle habituation experiments, rats exhibited a dose-related decrease in response amplitude and rate of habituation. In the day 21 females, caudate nucleus dopamine (DA) and serotonin (5-HT) concentrations and DA-receptor binding were decreased in a dose-dependent manner. Males showed less dramatic, but similar trends in caudate changes. However, hippocampal 5-HT and 5-HT receptor binding were significantly reduced only in females. Thus, sex-related behavioral alterations were accompanied by sex-related neurochemical changes, and females generally were more affected than males by prenatal reserpine treatment. The significant decrease in activity and auditory startle amplitude in the females is consistent with the suggested down regulation of the DA system in regional brain areas.

Transplacental estrogen responses in the fetal rat: increased uterine weight and ornithine decarboxylase activity.

The synthetic estrogens, diethylstilbestrol (DES) and ethynylestradiol (EE2), are more potent than 17 beta-estradiol (E2) in inducing uterine weight gain in the neonatal rat, due to the binding of E2 to serum alpha-fetoprotein (AFP). However, all three hormones are equipotent in inducing neonatal uterine ornithine decarboxylase (ODC) activity. The present study assessed estrogen potency in fetal rats. Pregnant CD rats were injected sc daily on gestation days (GD) 16-20 with DES, EE2, or E2 in sesame oil. Both DES and EE2, but not E2, significantly increased uterine weight at birth, to more than twice that of controls. In addition, implants which continuously release E2 only slightly increased uterine weight at birth. Alternatively, dams were given a single estrogen injection on GD 20 and were sacrificed at various times after injection. Peak fetal uterine ODC activity occurred at 6-8 hours after maternal injection for all three estrogens. E2 had a relative potency about tenfold less than either DES or EE2 in stimulating fetal ODC activity, in contrast to equal potencies of the three estrogens in the postnatal rat uterus. Similar patterns were found following direct fetal injection with E2 or DES. In summary, these data demonstrate a transplacental induction of fetal uterine ODC activity and uterine weight gain by both DES and EE2. In addition, the lack of correlation between these endpoints in response to E2 suggests that they may be useful as selective indicators of potential toxicity of both natural and synthetic estrogens.

Effect of prenatal reserpine exposure on development of the postnatal rat heart.

Previous studies have suggested that reserpine treatment may result in altered heart development. In order to more fully investigate this possibility, reserpine was administered s.c. at 0, 0.375, or 0.75 mg/kg/day to pregnant rats on gestation days 12-15. Maternal weight gain, as well as pup weight on postnatal day (PND) 1, was significantly reduced in a dose-dependent manner. Litter size was unaffected, but reserpine-treated dams had more dead pups than did control dams. On PND 1, litters were randomly standardized at ten pups each for analysis on PNDs 5, 8, 15, and 22. Pup body weight and heart weight were reduced in a dose-related manner at all ages measured. The decreased heart weights were probably due to decreases in cell number. Beta-adrenergic receptor concentration was significantly reduced only on PND 5, at the low reserpine dose, and was not considered to be a treatment effect. Prenatal reserpine exposure had no effect on levels of basal cardiac ornithine decarboxylase (ODC), an enzyme associated with growth and development. Cardiac ODC stimulation by insulin and isoproterenol also showed no effects of maternal reserpine treatment. The results suggest that maternal reserpine treatment may lead to adverse effects in the developing offspring.

Insulin stimulation of ornithine decarboxylase activity in developing rat heart.

Ornithine decarboxylase (ODC; EC 4.1.1.17) is an important enzyme in the synthesis of polyamines and is associated with growth and differentiation. Insulin stimulation of cardiac ODC has been proposed as a marker of the functional completion of the sympathetic pathway to the rat heart. However, earlier studies, using subsaturating substrate concentrations and a single time point measurement after insulin treatment, have been inconsistent concerning the postnatal age at which significant insulin stimulation of ODC occurs. The present study, using a validated near-saturating substrate assay, examines more thoroughly early neonatal insulin induction of cardiac ODC with respect to both the magnitude and the time course of response. Insulin (20 IU/kg s.c.) significantly increased ODC activity at several time points at each postnatal age measured (days 2, 5, 8, 15 and 22), with maximum ODC activity occurring by 2.5 to 3 hr after insulin injection at all ages. Insulin-stimulated ODC activity was increased over control levels by 86, 84, 87, 150 and 127% on days 2, 5, 8, 15 and 22, respectively. These results demonstrate that age is not a variable in the time of peak insulin stimulation of ODC activity and, in contrast to earlier reports, show that significant insulin induction of cardiac ODC activity occurs reliably across ages in the early postnatal period. The inconsistency of earlier studies may be due to a number of factors, including the use of subsaturating enzyme assays only, known to be subject to several types of error.

Effect of prenatal imipramine exposure on development of the postnatal rat heart and brain.

Imipramine (IMI) was administered s.c. at 0, 5, or 10 mg/kg/day to pregnant rats on gestation days 8-20 to assess possible alterations in postnatal heart and brain development. Maternal weight gain was significantly reduced in a dose-response manner, but litter size and pup weight on postnatal day (PND) 1 were unaffected. On PND 1, litters were culled to 10 pups for analysis on PNDs 4/5, 7/8, 14/15, and 21/22. Pup body weight was not affected at any age measured, but heart weight was significantly reduced at 10 mg/kg IMI on PNDs 4/5 and 7/8. Brain weight was increased in a dose-related pattern on PNDs 4/5 and 7/8 and was significantly higher at 5 mg/kg IMI on PND 14/15. No significant effect was observed in heart or brain protein and DNA content or in cardiac beta-adrenergic receptor concentration. Prenatal IMI exposure had no effect on basal cardiac ornithine decarboxylase (ODC), an enzyme associated with growth and development, but basal brain ODC was lower at 5 mg/kg IMI at all ages measured. Cardiac ODC stimulation by insulin was unaffected by prenatal exposure to IMI, but isoproterenol-stimulated ODC was increased on PND 21/22 at 5 mg/kg IMI. In conclusion, the IMI-related changes in several parameters suggest that when maternal IMI treatment is used, alterations in postnatal heart and brain development must be considered as possible outcomes.

Effect of prenatal propranolol exposure on development of the postnatal rat heart.

Maternal propranolol (PRO) treatment has previously been associated with adverse effects on the fetus and neonate. In the present study, pregnant rats were treated with PRO (25 or 50 mg/kg/day s.c.) on gestation days 8-20 to assess its possible effects on the developing heart. Maternal weight gain and pup weight on postnatal day (PND) 1 were reduced in a dose-dependent manner; litter size was unaffected. Pup body weight and heart weight both showed a dose-related decrease at all ages tested (PNDs 5/6, 8/9, 15/16, and 22/23). Since heart protein, but not DNA, was similarly reduced, the decrease seen in heart weight most likely reflects a decrease in cell size instead of cell number. Basal ornithine decarboxylase (ODC), an enzyme associated with growth and development, was unaffected by maternal PRO treatment. Insulin and isoproterenol stimulation of ODC, suggested markers for testing the function of the sympathetic pathway to the heart and of the heart's ODC response system, respectively, also showed no PRO-related response. In conclusion, prenatal PRO exposure resulted in reduced body weight, heart weight, and heart protein, but had little effect on heart DNA or ODC activity. Since PRO treatment also reduced maternal weight gain, the adverse effects seen in the pups may be due to generalized PRO toxicity. The results suggest that when high PRO doses were used clinically, the careful monitoring of maternal weight gain during pregnancy might be useful in predicting adverse fetal effects.

Ontogeny of cardiac ornithine decarboxylase and its beta adrenergic responsiveness in the rat.

Ornithine decarboxylase (ODC) is a marker of tissue growth and development and, because sympathetic stimulation of beta adrenergic receptors acutely increases ODC in the adult rat heart, measurement of this enzyme can be used to indicate the functional intactness of the beta adrenergic receptor system in the heart. Changes in the postnatal ontogenetic pattern of this enzymatic activity may also indicate abnormal development and ODC appears to be particularly useful in evaluating the effects of prenatal insult on cardiac development. The present study examines the pattern of basal ODC activity and its developing sensitivity to beta adrenergic stimulation during the perinatal period in order to establish a data base for studies on the effect of various environmental agents on the developing cardiovascular system. ODC activity was measured in rat hearts on gestation day (GD) 20 through postnatal day (PND) 28 under saturating conditions of L-ornithine and pyridoxal 5-phosphate. Basal ODC activity fell from 3 nmol of CO2/hr/mg of protein at GD 20 to less than 0.5 nmol of CO2/hr/mg of protein at PND 18, rising again to nearly 1 nmol of CO2/hr/mg of protein at PND 22. The beta adrenergic agonist isoproterenol (10 mg/kg s.c.) resulted in peak ODC stimulation at 4 hr postinjection on PNDs 6, 14 and 21; however, no response was seen at PND 1 at this dose or at GD 20 (300 micrograms/kg s.c.). In dose-response studies, isoproterenol produced a maximal response at 10 mg/kg s.c., resulting in increases from control of 67, 230 and 1700% at PNDs 6, 14 and 21, respectively, indicating that the sensitivity of the heart to beta adrenergic stimulation increases with age, during the perinatal period.

Characterization of estrogen binding in uterine cytosol from the fetal rhesus monkey.

Cytosol receptor binding of 17 beta-estradiol was demonstrated in the uterus of the late-gestation, fetal rhesus monkey. Sucrose density gradient analysis performed in low-ionic strength buffer indicated a binding component with a sedimentation coefficient of 6-7 S. Under high-ionic strength conditions, the component shifted to a sedimentation coefficient of approximately 4 S. The specificity of the receptor for estrogens was indicated by inhibition of [3H]estradiol binding by both natural and synthetic estrogen competitors, but not by progesterone. Saturation analysis indicated a high degree of nonspecific binding with saturation of specific binding occurring at 2-3 nM. Computer-assisted Scatchard analysis of the data resolved a one-receptor model having a limited number of binding sites and an apparent dissociation constant of 10(-10) M. The interaction of estrogens with the fetal uterus and the cellular mechanisms which permit this interaction are discussed in relation to the development of models for extrapolation to the human.

Barrier animal facility operations for a large-scale chronic study (ED01 study).

The physical facilities of the specific pathogen free/defined flora barrier animal rooms and their support operations are described as used in the conduct of a large-scale, long-term, low-dose carcinogenicity study. Specifically described are the floor plan of the facility, the mechanical operations, air flow, temperature and humidity controls, personnel screening and requirements, cleaning operations, and traffic flow patterns of personnel, equipment, and animals.

Estimation of the LD1 and extrapolation of the LD0.1 for five organophosphate pesticides.

The oral LD50,s for organophosphate pesticides have been determined in CD-1 strain male and female mice. The values in mg/kg are: Trichlorfon, 800 and 800; Naled, 409 and 330; Dichlorvos, 139 and 133, GC6506, 23.4 and 17.8; Fospirate, 225 and 263 respectively. Toxicity was greater in males with Fospirate and greater in females with Naled and GC6506. The predicted LD1's and the extrapolated LD0.1's have been determined for the 5 organophosphates from an unbalanced design, loaded heavily toward the lower end of the dose-response curve. It has been shown that the slopes of the curves obtained with 50, 100 and 660 animals are parallel for all compound except Fospirate in the 660 mouse experiments. This is probably related to excessive female deaths in the upper segment of the dose-response curve. Sex dependent lethality was observed with Trichlorfon, Dichlorvos and Fospirate with the males being more susceptible than the females except in the case of Fospirate where there was a reversal at the LD50 with greater susceptibility in the females. The conditions for obtaining accurate results in such experiments have been established. The implications of human exposure to low levels of the environmental pollutants have been discussed.

Estimation of the LD1 and extrapolation of the LD0.1 for five organothiophosphate pesticides.

The oral LD50's for five organothiophosphate pesticides have been determined in CD-1 strain male and female mice. The values in mg/kg are: parathion, 15.0 and 14.3; methyl parathion, 14.5 and 19.5; guthion, 7.15 and 6.35; imidan, 25.2 and 23.1; sumithion, 1,045 and 1,220 respectively. Toxicity was greater parathion but there was no difference in the males. The relationship between chemical structure and toxicity has been discussed. The addition of a methyl group in the meta position of the nitrophenyl group (sumithion) decreased toxicity 72 times compared to methyl parathion. The predicted LD1 and extrapolated LD0.1's have been determined for the five organothiophosphates and the conditions required for accurate results have been established. It has been shown that the slopes of the curves (males vs. females) obtained with 50, 100 and 660 animals are parallel for all compounds health implications of exposure to low levels of environmental pesticides have been discussed.