Two-generation reproduction studies in Rats fed di-isodecyl phthalate.

Di-isodecyl phthalate (DIDP) is a commercial plasticizer with low toxicity in many animal studies. The effects of dietary DIDP administration on fertility and developmental parameters were assessed in Sprague-Dawley rats utilizing two generation reproductive toxicity studies generally consistent with current regulatory guidelines. Dietary levels ranged from 0.02 to 0.8% (or approximately 15 to 600 mg/kg/day). In the reproductive studies, there were no effects on fertility, but there were decreases in adult body weight along with corresponding increases in liver and kidney weights and histopathologic changes indicative of peroxisomal proliferation. There were no effects on live birth index, but reduced offspring survival was observed at postnatal days 1 to 4. This reduced survival was more pronounced in the F2 generation in which statistical significance was achieved at levels of 0.2% DIDP and greater. There were also transient decreases in offspring body weights prior to weaning, corresponding to rapid offspring growth, and high levels of food consumption. There were no notable alterations in developmental landmarks. Overall, these studies provided experimentally defined No-Observed-Adverse-Effect Levels (NOAELs) of 0.06% (approximately 50 mg/kg/day) for F2 offspring survival and 0.8% (approximately 600 mg/kg/day) for fertility, other measures of reproductive function, and developmental landmarks. Statistical evaluation of the data from both studies identified 108 mg/kg/day with a 95% lower bound value of 86 mg/kg/day as a theoretical NOAEL for reduced F2 offspring survival.

Percutaneous pulmonary artery gene transfer in pigs using naked plasmid DNA delivered during angioplasty.

Gene transfer techniques are increasingly being used to study blood vessel biology and develop models for gene therapy. To date, there are no reports of pulmonary vascular gene transfer performed either without adjunctive agents or during angioplasty. We sought to demonstrate the feasibility of recombinant gene transfer to the pulmonary artery of juvenile pigs using naked plasmid DNA delivered via percutaneous angioplasty techniques. Plasmid DNA directing the expression of beta-galactosidase was used to transfect one pulmonary artery while the contralateral vessel served as an untreated control. One delivery technique used a standard angioplasty balloon coated with a DNA-heparin mixture. The second involved infusion of DNA between an angioplasty balloon and a surrounding, microporous balloon. Vessels were harvested 3 or 4 days following gene delivery. Protein expression was demonstrated by immunohistochemical staining in transfected but not control vessels in 9/9 pigs. Vascular wall expression was limited to endothelial cells. Pulmonary artery gene transfer using naked plasmid DNA delivered via percutaneous angioplasty techniques is feasible. Using naked plasmid DNA removes the potential for toxicity associated with adjunctive agents. The described techniques provide novel methods for studying pulmonary vascular biology in vivo and for developing future gene therapies.

Two-generation reproduction study in rats given di-isononyl phthalate in the diet.

The potential reproductive toxicity of di-isononyl phthalate (DINP: CAS RN 68515-48-0) was assessed in one- and two-generation reproductive toxicity studies. Groups of 30 male and female CRL : CD(SD)BR rats were given DINP via dietary administration at levels of either 0.0, 0.5, 1, or 1.5% (one-generation study) or 0.0, 0.2, 0. 4, or 0.8% (two-generation study). There were no changes in any of the classic reproductive parameters, i.e. mating, male or female fertility, fecundity, gestational index, or length of gestation in either study. The overall NOAELs for these effects were the highest Dietary Level (%)s tested, approximately 500 mg/kg/day in the two-generation study and 1000 mg/kg/day in the one-generation study. There were no testicular effects in parental animals exposed as juveniles and young adults at 960 mg/kg/day in the one-generation study. In the two-generation study, there were no testicular effects in either the P(1) males, exposed as juveniles and young adults or the P(2) (F(1)) offspring exposed in utero, through lactation, and continuously to terminal sacrifice. The NOAEL was 470 mg/kg/day. Offspring survival was reduced at the 1.5% level ( approximately 1100 mg/kg/day) but unaffected at the 1% level ( approximately 760 mg/kg/day). There were decreased offspring body weights both at postnatal day (PND) 0 and during lactation; however, the PND 0 effects were only clearly related to treatment at the 1.5% level. Weights of offspring during lactation were significantly reduced but within the historical control range at Dietary Level (%)s below 1%. As there was rapid recovery at the lower levels, even though treatment continued, the toxicologic significance is unclear. Adult survival was unaffected at any level in either study, but weight gain was significantly reduced at the 1% level ( approximately 600 mg/kg/day). Liver and kidney weights were elevated at Dietary Level (%)s above approximately 110 mg/kg/day, consistent with evidence from other studies of peroxisomal proliferation at these levels. This study showed that DINP treatment does not affect fertility or male reproductive development at doses of up to approximately 1000 mg/kg/day.

Evaluation of skin sensitization response of dialkyl (C6-C13) phthalate esters.

Isolated case reports suggest that dermal contact with some phthalate esters may result in skin sensitization. This issue was investigated in guinea pig sensitization tests, but the results were inconclusive. Consequently, 7 dialkyl phthalate esters, (diisohexyl, diisoheptyl, di(2-ethylhexyl), diisononyl, diisodecyl, diundecyl and ditridecyl phthalates), ranging in carbon number from C6 to C13, were tested in a 104-person panel human repeated insult patch test (HRIPT) using the modified Draize procedure. Test concentrations of 100% were selected for the induction and challenge phases of the HRIPT based upon a 24-h occluded irritation test on 15 panelists. Under the conditions of this HRIPT, no evidence of dermal irritation or sensitization for any of the 7 phthalate esters was observed in the 104-person panel. These HRIPT data provide evidence for the lack of experimental skin sensitization potential for the phthalate esters tested.

Developmental toxicity of di-isodecyl and di-isononyl phthalates in rats.

The developmental toxicity of di-isodecyl phthalate (DIDP; CAS RN 68515-49-1) and di-isononyl phthalate (DINP; CAS RN 68515-48-0) were investigated in Sprague-Dawley rats. DIDP and DINP were administered by gavage to mated rats at doses of 0, 100, 500, and 1000 mg/kg/d on Gestation Days (GD) 6 through 15. Cesarean sections were performed on GD 21 and the fetuses removed for evaluation. Maternal weight gain and food consumption were significantly reduced at 1000 mg/kg/d during the exposure period. No treatment-related effects were noted at cesarean section, nor were there any fetal morphologic observations except for an increased frequency of seventh cervical and rudimentary lumbar ribs at the maternally toxic exposure level of 1000 mg/kg/d. Under these study conditions, mild maternal and developmental effects were observed at 1000 mg/kg/d. Both maternal and developmental NOAELs were therefore established at 500 mg/kg/d. The results indicate that neither DIDP nor DINP is teratogenic or a selective developmental toxicant.

Developmental toxicity assessment of C8 iso acid in CD rats: relevance to embryotoxic aliphatic carboxylic acids.

Cekanoic C8 acid (CAS RN 25103-52-0) is a complex isomeric substance containing several aliphatic carboxylic acids, primarily dimethyl hexanoic acid. Because Cekanoic C8 acid is a structural isomer of octanoic acid, its potential for developmental toxicity was investigated in CD (Sprague-Dawley) rats. Cekanoic C8 acid was administered by oral gavage to 25 confirmed-mated females at doses of 0, 200, 400, and 800 mg/kg/day on gestation days (GD) 6-15, based on a range-finding experiment. Maternal body weights, food consumption, and clinical observations were recorded throughout gestation. On GD 21, cesarean sections were performed and the uterine contents removed and subjected to conventional teratological evaluation. At 800 mg/kg/day, maternal body weight gain and food consumption were reduced during the exposure period, and clinical signs were evident. There were no significant differences in fetal weight, malformation incidence, or fetal viability in any of the experimental groups. There was a statistically significant increase in the incidence of total variations in the 800-mg/kg/day group, which was within the historical control range of this laboratory and not considered biologically significant. These results indicate that, unlike related compounds, Cekanoic C8 acid was not teratogenic or a selective developmental toxicant in rats. This is the first report of a dimethyl substituted aliphatic acid being evaluated for developmental toxicity in a definitive study. The results are consistent with a structure-teratogenicity relationship for aliphatic acids, indicating that side-chain branching larger than a methyl group is required to elicit teratogenic effects. This study established a maternal no-observable-adverse-effect level (NOAEL) for Cekanoic C8 acid at 400 mg/kg/day and a developmental NOAEL at 800 mg/kg/day.

Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression.

A series of vectors has been developed to provide improved positive and negative selection for allelic exchange. Based on homologous regions of DNA ranging in size from less than 200 bp to over 1 kb, we have successfully used these new plasmids to introduce or remove markers in chromosomal or plasmid DNA. Wild type fimbria genes were replaced both in Salmonella enteritidis (sefA, agfA and fimC) and Escherichia coli (fasA and fasH). Regulation of 987P fimbriation could be identified after replacement of fasA and fasH with allelic reporter fusions. The expression of fasA but not fasH is dependent upon the osmolarity of the growth medium in an HNS-dependent manner, but unlike some other fimbrial systems expression is not dependent on the exogenous iron concentration.

Invasion of chicken reproductive tissues and forming eggs is not unique to Salmonella enteritidis.

Experiments were conducted in which Salmonella enteritidis Phage Type 8, Phage Type 2, and RDNC (reaction does not conform) or three isolates of Salmonella typhimurium of diverse origin were fed to adult laying hens to determine if S. enteritidis has a selective advantage over S. typhimurium, which is now rarely isolated from chicken eggs, in its capacity to invade reproductive tissues. The results revealed that S. enteritidis and S. typhimurium may be equal in their potential to colonize the tissues of the reproductive tract and eggs that are forming in the oviduct prior to oviposition. S. enteritidis, but not S. typhimurium, was isolated from egg contents after oviposition. The degree to which intestinal, hepatic, splenic, or reproductive tissues were colonized by either serotype was not seen to affect the rate of colonization of eggs forming in the oviduct or the contamination of eggs after oviposition. Virulence factors related to the difference in the association of S. enteritidis and S. typhimurium with egg-borne salmonellosis remain to be defined.

A novel relationship between O-antigen variation, matrix formation, and invasiveness of Salmonella enteritidis.

Salmonella enterica Enteritidis in chickens serves as a reservoir for salmonellosis in humans and the structure of its lipopolysaccharide (LPS) has been used to assess invasiveness. Culture from chick spleens generated colonies with an unusual wrinkled morphology, and it is designated the lacy phenotype. The characterize the nature of the morphological change, three isogenic variants were compared. Only the lacy phenotype produced a temperature-dependent cell surface matrix composed of several proteins in association with LPS high molecular weight O-antigen. Flagellin and a 35 kDa protein were identified as specific proteinaceous components of matrix. Both proteins cross-reacted with a monoclonal antibody previously determined to specifically detect the g-epitope of the Enteritidis monophasic flagella (H-antigen). These results suggest that O-antigen in association with protein contributes to cross-reactivity between molecules. The lacy phenotype was more organ invasive in 5-day-old chicks than isogenic variants producing low molecular weight O-antigen. However, it was no more efficient at contaminating eggs after oral inoculation of hens than a variant that completely lacked O-antigen, thus the lacy phenotype is classified as an intermediately invasive organism. The distinctive colonial phenotype of SE6-E21lacy was used to investigate environmental factors that decreased O/C ratios and contributed to attenuation. In so doing, it was found that growth in complement at 46 degrees C caused matrix producing cells to hyperflagellate and migrate across agar surfaces. These results suggest that the structure of O-antigen might influence the secretion and/or the function of Enteritidis cell-surface proteins. The data also reveal a greater heterogeneity than has been assumed in the phenotype, and possibly the infectious behaviour, of Enteritidis.

Alkyl polychlorinated dibenzofurans and related compounds as antiestrogens in the female rat uterus: structure-activity studies.

The antiestrogenic activity of a series of alkyl-substituted polychlorinated dibenzofurans (PCDFs) were determined in the immature female Sprague-Dawley rat uterus. The compounds utilized in this study contain two, three, or four lateral substitutents and include: 6-methyl-1,3,8-triCDF, 6-ethyl-1,3,8-triCDF, 6-n-propyl-1,3,8-triCDF, 6-i-propyl-1,3,8-triCDF, 6-t-butyl-1,3,8-triCDF, 8-methyl-1,3,6-triCDF (two lateral substituents); 6-methyl-2,3,8-triCDF, 6-methyl-2,3,4,8-tetraCDF, 8-methyl-1,3,7-triCDF, and 8-methyl-1,2,4,7-tetraCDF (three lateral substituents); 8-methyl-2,3,7-triCDF, 8-methyl-2,3,4,7-tetraCDF (four lateral substituents). Two additional compounds, 8-methyl-2,3,7-trichlorodibenzo-p-dioxin and 8-methyl-2,3,7-tribromodibenzo-p- dioxin (four lateral substituents), were also utilized. All of the alkyl-substituted compounds inhibited estrogen-induced uterine wet weight increase and cytosolic and nuclear progesterone and estrogen receptor binding. The effects of structure on the antiestrogenic potencies were determined using 6-i-propyl-1,3,8-triCDF, 6-methyl-2,3,4,8-tetraCDF, and 8-methyl-2,3,4,7-tetraCDF as representative congeners containing two, three, and four lateral substituents, respectively. The ED50 values for antiestrogenicity were similar for the three compounds; however, the ED50 values for induction of hepatic CYP1A1-dependent activity were 73,600 (estimated), 8.52, and 5.31 mumol/kg for 6-i-propyl-1,3,8-triCDF, 6-methyl-2,3,4,8-tetraCDF, and 8-methyl-2,3,4,7-tetraCDF, respectively. Since CYP1A1 can be used as a surrogate for toxic potency in the rat then high ED50 (induction)/ED50 (antiestrogenicity) ratios would be indicative of low toxicity and high antiestrogenic potency. The ratio was 13,990 to 17,100 for 6-i-propyl-1,3,8-triCDF, whereas the corresponding value for the compounds with three or four lateral substituents varied from 0.64 to 3.34. The results suggests that the 1,3,6,8-substituted alkyl PCDFs are useful structural models for developing new aryl hydrocarbon receptor-mediated antiestrogens for future clinical use as antiestrogens.

Salmonella enteritidis colonization of the reproductive tract and forming and freshly laid eggs of chickens.

Salmonella enteritidis colonizes the tissues of the chicken ovary and oviduct, presumably contaminating eggs and thereby contributing to human outbreaks of salmonellosis. In this study, commercial adult laying hens were given an oral inoculation of 10(8) S. enteritidis organisms. Tissues from various organs, the intestines, and the reproductive tract, including freshly laid eggs, were collected daily for up to 40 days postinoculation (p.i.). Within 2 days p.i. S. enteritidis was detected by culture in pools of the spleen, liver, heart, and gallbladder tissues, in intestinal tissues of all infected birds, and in various sections of the ovary and oviduct. Detection of organisms by immunohistochemical staining was rare for most tissues in spite of their culture-positive status, suggesting a low level of tissue colonization. However, S. enteritidis could be detected by immunohistochemical staining in oviduct tissues associated with four forming eggs, indicating the possibility of a heavier colonization in the egg during its development. In two subsequent experiments, forming eggs taken from the oviduct with their associated tissue, were found to be culture positive for S. enteritidis at a rate of 27.1 and 31.4%, while freshly laid eggs in these experiments were culture positive at the rate of 0 and 0.6%. These observations suggest that while forming eggs are significantly colonized in the reproductive tract, factors within the eggs may control the pathogen before the eggs are laid. The data show that prior to egg deposition, forming eggs are subject to descending infections from colonized ovarian tissue, ascending infections from colonized vaginal and cloacal tissues, and lateral infections from colonized upper oviduct tissues. The data are consistent with an ascending infection of freshly laid eggs from the cloaca, as the incidence of positive eggs in experiments 1 and 3 coincided with heavily contaminated cloacal tissues (50.7 and 80%, respectively), while no positive eggs were detected in experiment 2 when cloacal colonization was low (8.3%). The data do not support the possibility of egg invasion by bacterial translocation from the peritoneal cavity.

Monoclonal antibody-based detection system for Salmonella enteritidis.

Two monoclonal antibodies that react with Salmonella enteritidis in chicken tissue, eggs, and environmental samples were used to develop a rapid enzyme-linked immunosorbent assay (ELISA) screen and agglutination assay for the specific detection of S. enteritidis. S. enteritidis was detected in 100% of egg samples and 99.8% of various field and research samples by both ELISA and traditional microbiological isolation and identification techniques. Results of titer experiments indicate that as few as 10(4) organisms can be detected by ELISA.

Protection against reticuloendotheliosis virus-strain T tumours is associated with JMV-1 culture supernatant-enhanced natural killer cell activity.

One-day-old chicks were inoculated intraperitoneally with cell-free JMV-1 culture supernatant and were subsequently challenged with a lethal dose of RECC-CU60 tumour cells at either 7 or 12 days of age. A significant reduction in mortality was evidenced in chicks that had been inoculated with JMV-1 culture supernatant prior to RECC-CU60 tumour cell challenge at 12 days of age, compared with all other treatment groups. Reducing the serum content of JMV-1 culture medium or changing the base medium in which the JMV-1 cells were grown did not affect the reduction in mortality, demonstrating that the protective effects against RECC-CU60 tumour cell challenge were a result of factors secreted into the culture medium by JMV-1 cells. Development of natural killer (NK) cell activity correlated with findings in the RECC-CU60 tumour cell challenge studies. Inoculation of 1-day-old chicks with JMV-1 culture supernatant resulted in a significant increase in NK cell activity in spleens taken from JMV-1 inoculated chicks 12 days later, when compared with spleens taken from chicks in all other treatment groups. Inoculation of chicks with RECC-CU60 tumour cells caused a suppression of their NK cell activity. Treatment of chicks with JMV-1 supernatant at day of age, prior to RECC-CU60 tumour cell challenge restored and significantly enhanced their NK cell activity. These results suggest that the JMV-1 cell line secretes lymphokines that protect against RECC-CU60 tumour cell challenge by directly or indirectly stimulating NK cell activity.

Differences among restriction endonuclease DNA fingerprints of Pennsylvania field isolates, vaccine strains, and challenge strains of infectious laryngotracheitis virus.

Restriction endonuclease fingerprints of infectious laryngotracheitis virus (ILTV) DNA from 13 Pennsylvania field isolates, embryo-propagated and tissue-culture-propagated vaccine strains, and three reference strains were compared. These comparisons were made to evaluate the possible contribution of mutation of ILTV vaccine strains to recent outbreaks of infectious laryngotracheitis (ILT) in Pennsylvania. Six different restriction enzymes were used to generate the fingerprints. Differences in DNA banding patterns were revealed between the currently used ILTV vaccine strains and six of the 13 field isolates. Even greater DNA banding pattern differences were found between the older ILTV reference strains and the vaccine strains. The ILTV DNA fingerprints generated in the present study suggest that at least five different strains of ILTV have contributed to the outbreaks of ILT that have occurred since 1987 in Pennsylvania.

JMV-1 stimulation of avian natural killer cell activity.

The phenotypic characterization of the non-productive, Marek's disease virus-transformed, lymphoblastoid cell line, JMV-1 and the immunological effects of its cell-free culture supernatant were examined. It was verified that the JMV-1 cell line is an activated T-helper cell line that bears antigens indicative of T-cell activation, and it is not a natural killer or cytotoxic T-cell line. Furthermore, the JMV-1 cell line was shown to be unique among Marek's disease-transformed cell lines in that JMV-1 cells secrete factors that are able to stimulate in vitro natural killer cell cytotoxicity against the avian tumour cell line, LSCC-RP9, in a 4-h assay. Culturing spleen cells in the presence of JMV-1 supernatant for 4 h stimulates a significant increase in NK cell populations and in la-bearing cell populations, as detected by monoclonal antibody staining. These findings suggest that the protective effects against the parasitic and virally-induced lymphoproliferative poultry diseases, which have been credited to the JMV-1 cell line and to its cell-free culture supernatant, are in part due to lymphokine activation of NK cell cytotoxicity.

Studies of histocompatibility and immune response of chickens selected for resistance and susceptibility to Marek's disease.

Two sets (four lines) of chickens, lines 7 and 6 and lines P and N, the former of each set susceptible and the latter resistant to Marek's disease, were examined for their relative histocompatibility and immunocompetence. Results from the in vivo graft-versus-host response splenomegaly assay, and graft-versus-host chorioallantoic membrane pock formation assay confirmed the within-line, B-locus homozygosity of chickens of lines 7, 6, and N and the heterozygosity of line-P chickens. These assays further confirmed that line-7 and line-6 chickens share identical alleles at the major histocompatibility locus. The capacity of the lines of chickens to elicit specific cell-mediated immune lysis as measured by the release of chromium 51 generally agreed with the in vivo graft-versus-host responses. These data demonstrate that the 51Cr-release assay is a reliable measure of histocompatibility within the avian system.

Isolation and development of a reticuloendotheliosis virus-transformed lymphoblastoid cell line from chicken spleen cells.

The establishment and characterization of a reticuloendotheliosis virus (strain T)-transformed lymphoblastoid cell line, designated TV-1, was reported. These cells, isolated from the spleen of a moribund chick infected with reticuloendotheliosis virus, were maintained in suspension culture for over 74 weeks at a stable generation time of 15 h. The cells were found to carry a female karyotype. Both TV-1 cells and cell-free TV-1 culture supernatant produced lesions and mortality patterns in chicks which were identical to those caused by reticuloendotheliosis virus (strain T) infection. Examination of TV-1 cells by electron microscope revealed the presence of C-type virions budding from the plasma membrane. Cytotoxicity assays and fluorescent antibody tests indicated the presence of B-cell determinants on the TV-1 cell surface membrane.