Histological and finite element analysis of cell death due to irreversible electroporation.

Irreversible electroporation (IRE) has been shown to be an effective method of killing cells locally. In contrast to radiofrequency ablation, the mechanism by which cells are thought to die via IRE is the creation of pores in cell membranes, without substantial increase in tissue temperature. To determine the degree to which cell death is non-thermal, we evaluated IRE in porcine hepatocytes in vivo. Using pulse widths of 10 µs, bursts of 3 kV square-wave pulses were applied through a custom probe to the liver of an anesthetized pig. Affected tissue was evaluated histologically via stainings of hematoxylin & eosin (H&E), nitroblue tetrazolium (NBT) to monitor cell respiration and TUNEL to gauge apoptosis. Temperature was measured during the application of electroporation, and heat transfer was modeled via finite element analysis. Cell death was calculated via Arrhenius kinetics. Four distinct zones were observed within the ring return electrode; heat-fixed tissue, coagulation, necrotic, and viable. The Arrhenius damage integral estimated complete cell death only in the first zone, where the temperature exceeded 70°C, and partial or no cell death in the other zones, where maximum temperature was approximately 45°C. Except for a limited area near the electrode tip, cell death in IRE is predominantly due to a non-thermal mechanism.

The effects of feed restriction during in utero and postnatal development in rats.

This study determined the effects of feed restriction (FR) during in utero and postnatal life on standard reproductive toxicity and developmental immunotoxicity end points. Groups of 26 time-mated CD rats were fed various amounts of Purina 5002 diet from gestation day 7 through lactation. Control rats were fed once per day in amounts based on historical control feed consumption data, while the amounts fed to the FR groups were reduced by 10% (10% FR), 30% (30% FR), or 50% (50% FR) relative to controls. Selected F1 weanlings were necropsied on postnatal day (PND) 22, assessed for immunotoxicity end points between PND 22 and 27 or PND 52 and 56, or maintained on FR through PND 70. Thereafter, half the remaining F1 rats in each group were fed ad lib (recovery subgroup), while the rest continued on FR. Both subgroups were necropsied at 21 weeks of age. In the 10% FR group, slight decreases in maternal body weight had no effect on F1 offspring body weights, but did decrease F1 liver weights. FR at the 30% level reduced maternal body weights by 10-20%, reduced F1 offspring body weights by as much as 21%, caused changes in numerous weanling organ weights, but did not affect reproductive or immune system function. Dams in the 50% FR group were 17-32% lighter than controls, resulting in F1 body weights that were 12-47% lower than controls. F1 estrous cycle length was increased, puberty was delayed by 6 days (males and females), and anogenital distance, epididymal sperm counts, and all organ weights were decreased in this group. Antibody responses were unaffected despite decreased spleen and thymus weights. Essentially all effects of feed restriction showed evidence of reversibility.

Evaluation of the male pubertal onset assay to detect testosterone and steroid biosynthesis inhibitors in CD rats.

The male pubertal onset assay has been recommended by the Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) as an alternate Tier I screening assay to detect potential endocrine-active chemicals (EACs). Recently, this assay was evaluated by several laboratories using a variety of dosing schemes. This study used a 30-day dosing period to confirm and extend previous work on the assay's ability to detect steroid biosynthesis inhibitors. Weanling male rats were dosed by gavage from 21 to 50 days of age with vehicle (0.5% methocel) or chemicals from the following EAC classes: an androgen (testosterone propionate [TP], 0.1 or 0.4 mg/kg/day), a broad-spectrum steroid biosynthesis inhibitor (ketoconazole [KETO], 24 mg/kg/day), a 5alpha-reductase inhibitor (finasteride [FIN], 20 or 80 mg/kg/day), a moderately specific aromatase inhibitor (testolactone [TL], 220 mg/kg/day), or a highly specific aromatase inhibitor (fadrozole [FAD], 0.6 or 6.0 mg/kg/day). None of these treatments altered relative thyroid weights. However, TL, KETO, and FIN were positive for endocrine activity based on decreases in one or more reproductive or accessory sex gland organ weights. Of these three inhibitors, only TL significantly increased the age at PPS, indicating that PPS was less sensitive for detecting these EACs. Based on its profile of effects, TL may have been detected as an antiandrogen. TP and FAD were negative in this assay, even at doses that caused effects in other studies. With TP, oral administration limited assay sensitivity such that higher TP doses would be needed for detection. FAD decreased body weight gains, but did not significantly alter any other assay end points; thus, the capacity of this assay to detect aromatase inhibitors remains in question.

Evaluation of the male pubertal assay's ability to detect thyroid inhibitors and dopaminergic agents.

The male pubertal onset assay is under consideration as an alternate Tier I screening assay to detect potential endocrine active chemicals (EACs) acting through a variety of steroid hormone and thyroid hormone receptor-mediated and non-receptor-mediated mechanisms. This study focused on the assay's ability to detect several non-receptor-mediated EACs. Weanling male CD rats (21 days old) were dosed for 30 d by gavage with vehicle (0.5% METHOCEL) or the following EAC classes (mg/kg/d): a potent thyroid agent (6-propylthiouracil, PTU, 240), a weak thyroid agent (phenobarbital, PB, 50 or 100), a dopamine antagonist (haloperidol, HALO, 2 or 4), or a dopamine agonist (bromocryptine, BRC, 10 or 50). In vehicle-treated males, preputial separation (PPS) occurred at 44.4 +/- 2.0 days of age. Age at PPS was delayed with PTU and 50 BRC, treatments that also delayed growth. Absolute testes and/or epididymal weights were decreased by PTU and 100 PB. BRC (50) and PB (100) decreased absolute prostate and seminal vesicle weights. Relative thyroid weights were altered by HALO, PTU, and PB, agents that significantly decreased serum T(4) levels. PTU increased serum thyroid-stimulating hormone (TSH) by 8.5 times and markedly altered thyroid histology, whereas HALO and PB did not significantly increase TSH and had marginal effects on thyroid histology. Thus, this assay detected both strong (PTU) and weak (PB) thyroid agents as well as the dopamine agonist BRC; however, its ability to detect dopamine antagonists remains unproven. These results confirm that thyroid weight measurements, although not required in the current male pubertal assay protocol, may add valuable information for interpretation of thyroid effects. Due to the apical nature of the male pubertal assay end points, additional work will be required to establish definitive criteria for a positive result in this assay.

Postulated human sperm count decline may involve historic elimination of juvenile iodine deficiency: a new hypothesis with experimental evidence in the rat.

Human sperm count studies, historic dietary iodination, and an animal model where neonatal goitrogen administration causes unprecedented testis enlargement, together suggest an hypothesis relevant to the postulated fall in human sperm counts. We present the hypothesis with a supporting study extending the model to include iodine deficiency. In a one-generation rat reproduction study, dams were fed an iodine sufficient (control, 200 ppb I) or deficient (low iodine diet [LID], <20 ppb I) diet from prebreeding through weaning, when male offspring were divided into three groups: 1) controls from iodine sufficient dams, 2) neonatal LID (NLID) from the LID dams, fed control diet postweaning, and 3) chronic LID (CLID) from LID dams, fed a moderate LID (40 ppb I) postweaning. F1 males were euthanized on postnatal day (PND) 133+/-1. Each of the three diet groups comprised two subgroups in which testicular parameters were evaluated: 1) daily sperm production (DSP), sperm motility, morphology, and histopathology, and 2) Sertoli cell and round spermatid morphometry. In the first subgroup, NLID and CLID testes weights were 8.5% and 14.0% heavier than their unusually heavy controls (3.921 g; historical control mean approximately 3.5 g), with proportional DSP increases. Sperm motility, morphology, and testis histopathology were unaffected. In the morphometry subgroup, respective increases in NLID and CLID rats included testes weights (+28.6% and +20.3%), Sertoli cells (+24.8% and +23.9%), and round spermatids (+20.4% and +15.8%). The results indicate that neonatal iodine deficiency can significantly increase spermatogenic function in rats, and support our hypothesis concerning human sperm counts.

Assessment of adult and neonatal reproductive parameters in Sprague-Dawley rats exposed to propylene glycol monomethyl ether vapors for two generations.

This study evaluated propylene glycol monomethyl ether (PGME) in a rat 2-generation reproduction study, which included non-traditional study end points, such as sperm count and motility, developmental landmarks, estrous cyclicity, and weanling organ weights. Groups of 30 male and 30 female Sprague-Dawley rats (6-weeks-old) were exposed to 0, 300, 1000, or 3000 ppm of PGME vapors via inhalation for 6 hours/day, 5 days/week prior to mating, and 6 hours/day, 7 days/week during mating, gestation, and lactation, for 2 generations. These concentrations corresponded to estimated oral equivalent doses of 0, 396, 1325, or 3974 mg/kg/day. At 3000 ppm, toxicity in the P1 and P2 adults was marked, as evidenced by sedation during and after exposure, and mean body weights which were as much as 21% lower than controls. This marked parental toxicity was accompanied by lengthened estrous cycles, decreased fertility, decreased ovary weights, and histologic ovarian atrophy in maternal rats. In the offspring from these dams, decreased body weights, reduced survival and litter size, slight delays in puberty onset, and histologic changes in liver and thymus in the F1 and F2 offspring were observed. The nature of the reproductive/neonatal effects and their close individual animal correlation with decreased maternal body weights suggested that these effects were secondary to general toxicity and/or nutritional stress. No such reproductive/neonatal effects were observed at 1000 ppm, a concentration which caused less marked, but significant body weight effects without sedation. There were no treatment-related effects of any kind noted at 300 ppm of PGME. Therefore, the no-observable-effect level (NOEL) for reproductive/neonatal effects was 1000 ppm, and that for parental toxicity was 300 ppm.

Evaluation of the EDSTAC female pubertal assay in CD rats using 17beta-estradiol, steroid biosynthesis inhibitors, and a thyroid inhibitor.

The Endocrine Disrupter Screening and Testing Advisory Committee has recommended the female pubertal onset assay as a Tier I test to detect potential endocrine-disrupting chemicals (EDs). We evaluated this assay's ability to detect EDs acting through various mechanisms. In two similar experiments, weanling female rats were dosed for 20 days by gavage with vehicle (0.5% methocel) or the following test compounds (mg/kg/day): 17beta-estradiol (E2; 0.1, 2, or 4), ketoconazole (KETO; 24, 50, or 100), finasteride (FIN; 20), testolactone (TL; 220), fadrozole (FAD; 0.6, 1.2, or 6.0) or 6-propylthiouracil (PTU; 240). In vehicle-treated females, mean age at pubertal onset, as evidenced by vaginal opening (VO), varied interexperimentally from 32.3+/-1.6 days to 33.5+/-1.8 days. At 0.1 mg/kg E2, age at VO was reduced slightly to 31.0+/-1.6 days, but not significantly (alpha=0.05). Higher E2 doses (2.0 and 4.0) reduced age at VO to 28 days. KETO delayed VO, but this delay was significant only at 100 mg/kg (39.7+/-2.4 days). FIN and TL had no effect on age at pubertal onset; however, FAD significantly delayed VO. PTU delayed VO to 34.2+/-1.1 days and altered thyroid weight, histology, and hormone levels. With each compound, significant changes in age at VO were accompanied by decreased uterine or ovarian weights. Thus, although this assay did not detect TL or lower doses of E2 (0.1 mg/kg) or KETO (< or = 50 mg/kg), it was capable of detecting EDs operating through a variety of mechanisms.

In-situ self-assembling protein polymer gel systems for administration, delivery, and release of drugs.

Sequential block copolymers consisting of tandem repetition of amino acids have been constructed and genetically produced based on the natural repeating structures of silk and elastin protein. Combinations of silklike and elastinlike amino acid sequence blocks in a high molecular weight protein polymer are used to confer properties similar to those observed with hard block and soft block segmented polyurethanes. A certain subset of these silk-elastinlike protein compositions, termed ProLastins, will undergo an irreversible solution to gel transition in physiological, aqueous solution. The transition occurs over time and can be controlled by temperature, solution conditions, and additives which either prevent or promote hydrogen bond-mediated chain crystallization. The process involves no covalent crosslinking. Characterization of the gelling properties of various ProLastin compositions and their ability to release compounds which are incorporated directly into the gels are presented.

Single-dose and 13-week repeated-dose neurotoxicity screening studies of chlorpyrifos insecticide.

Chlorpyrifos (CPF), a widely used organophosphate insecticide, was screened for neurotoxic effects in Fischer 344 rats using United States Environmental Protection Agency 1991 guidelines for single-dose and 13-wk repeated dose studies. The studies emphasized a functional observational battery (which included grip performance and hindlimb splay tests), automated motor activity testing and comprehensive neurohistopathology of perfused tissues. Doses of up to 100 mg/kg body weight in corn oil by gavage in the single-dose study and up to 15 mg/kg body weight/day in diet for 13 wk in the repeated dose study were administered. It is known that CPF and other phosphorothionates can be activated to the oxon in local (extrahepatic) tissues. Local activation could possibly cause different effects in different tissues with cholinergic innervation, and thereby create syndromes unique to each phosphorothionate according to their structure. Consequently, the conduct of CPF neurotoxicity screening studies by contemporary guidelines offered opportunity to characterize the CPF over-exposure syndrome in rats. Single-dose high levels of oral exposure to CPF caused a range of clinical signs characteristic of cholinergic overstimulation. Although there was no clinical evidence of wide differences in sensitivity of one cholinergic response versus another, motor dysfunction (incoordination etc.) was more prominent than other signs, for example soiling. Effects were much more apparent in females and regressed over several days. Effects were minimal in the 13-wk study, and there was no evidence of accumulation of toxicity during the 13 wk of daily dietary exposure. Motor activity was decreased at the high dose in males and females at wk 4, but was not significantly different from controls in subsequent weeks. The 'normalization' of motor activity later in the study was interpreted as tolerance to repeated administration of CPF. Comprehensive neuropathological examination revealed no treatment-related lesions in either study.

Atypical tubule hyperplasia and renal tubule tumors in conventional rats on 90-day toxicity studies.

Bilateral, multicentric renal tubule tumors were found in 4 rats at the termination of 3 separate 90-day toxicity studies during the safety evaluation of 3 unrelated chemicals. The 3 studies were conducted at 2 separate locations, but the rats used were obtained from the same commercial source. The rat strains were Fischer-344 (1 male and 1 female case) and Sprague-Dawley (2 female cases). Three of the renal tumor cases were from either the high-dose or mid-dose treatment groups, and 1 case was an untreated control. The tumors were accompanied by multiple foci of atypical tubule hyperplasia but only in the tumor-bearing rats. There were no lesions associated with renal tumor pathogenesis in any of the remaining treated or untreated animals in the 3 studies. In addition, there was no indication of nephrotoxicity in the treated or untreated animals. Tumor morphology was characterized by a generally vacuolated appearance, eosinophilia, cytoplasmic and nuclear pleomorphism, and conspicuously hypertrophied nucleoli. The renal tubule tumors in these 90-day studies were compared to hereditary renal tubule tumors occurring in the Eker rat, a Long-Evans derivative with a genetic predisposition to this tumor type. The multiplicity of renal tubule tumors, early age of onset, and tumor morphology described in the cases from the 90-day studies were very similar to those characterizing the hereditary renal tumor model.(ABSTRACT TRUNCATED AT 250 WORDS)

Ganglioneuromas of the thyroid gland in a colony of Sprague-Dawley rats.

Ganglioneuromas of the thyroid gland of Sprague-Dawley rats were found in 7.2% of 698 rats used in two, 2-year oncogenicity bioassays. The incidence of the tumor was unrelated to treatment or sex. Of the 52 ganglioneuromas of the thyroid gland identified in 50 animals, 40 (80%) had coincident C cell proliferations in the same or contralateral lobe. Ganglioneuromas were contiguous or commingled with C cell proliferations in 63.5% of the cases. The ganglioneuromas consisted of large ganglion cells and, in a few cases, cells thought to be less differentiated neuronal precursors, in a matrix of neurites and Schwann cells. They grew, infiltrating and expanding in the thyroid parenchyma, and did not metastasize. Immunohistochemical staining for calcitonin, S-100 protein, and neurofilaments, as well as electron microscopy, were used to further characterize the tumors. The close association of ganglioneuromas with C cell tumors supports the theory of neural crest origin of C cells and provides a parallel to the association of ganglioneuromas and pheochromocytomas in the adrenal medulla. This is the first report of ganglioneuromas occurring in the thyroid gland or occurring as a common entity in any species.

Accumulation of the insecticidal crystal protein of Bacillus thuringiensis subsp. kurstaki in post-exponential-phase Bacillus subtilis.

A gene from Bacillus thuringiensis subsp. kurstaki that codes for a Lepidoptera-specific insecticidal toxin (delta-endotoxin) was engineered for expression in Bacillus subtilis. A low-copy-number plasmid vector that replicates in Escherichia coli and B. subtilis was constructed to transform B. subtilis with gene fusions first isolated and characterized in E. coli. Naturally occurring promoter sequences from B. subtilis (43, veg, ctc, and spoVG) were inserted upstream from the plasmid-borne structural gene. In the most prolific case, when the sporulation-specific spoVG promoter was fused to the heterologous toxin gene, the toxin product accumulated during postexponential growth to greater than 25% of the total cell protein. However, the resulting specific activity of the insecticidal toxin product was not commensurate with the abundance of the protein.

Morphologic stomach findings in rats and mice treated with the H2 receptor antagonists, ICI 125,211 and ICI 162,846.

The gross and histopathologic findings seen in the stomachs of rats and mice treated with 2 different H2 receptor antagonists are presented. Studies with the first drug, ICI 125,211, elicited dysplasia/carcinoma lesions in 17 of 828 treated rats with 12 of the 17 lesions occurring in the pyloric region of the stomach. No tumors occurred in mice on study for 18 months. Studies conducted with another drug candidate, ICI 162,846, produced neuroendocrine carcinomas in the stomach of rats and mice. Twenty-five rats out of 312 treated male and female rats had neuroendocrine carcinomas in the gastric fundus with a higher tumor incidence in females. In a 24-month mouse study with ICI 162,846, 45 of 300 treated mice developed neuroendocrine carcinomas in the gastric fundus with a higher incidence in males.

Regrowth of the rabbit meniscus after lateral and medial meniscectomies.

The rate of regrowth of the meniscus was compared after medial and lateral meniscectomies. Articular cartilage ulcers were observed 21 days after lateral meniscectomies and femoral pitting was observed after either procedure. Histologically, lesions were observed only after the lateral procedure. Regrowth of the meniscus was evident within 1 week of surgery as increased 3H-thymidine uptake and increased collagen synthesis by meniscal tissue from both surgical groups. These parameters returned to normal sooner after the medial than the lateral procedure. The excised meniscus was completely replaced by new tissue within 3 weeks of either procedure. In conclusion, the meniscus heals quickly following surgery and the small differences in the rate of regrowth between the two procedures do not seem to be adequate to explain the significant differences in femoral and tibial lesions.

Gene-III protein of filamentous phages: evidence for a carboxyl-terminal domain with a role in morphogenesis.

A filamentous phage derivative, fCA55, bearing a nonpolar deletion in gene III, has been constructed and characterized to study the functions of that gene. The deletion eliminates most of gene III without disturbing its reading frame or the putative promoter for the downstream gene, VI. Therefore it is assumed that any abnormalities exhibited by fCA55 are a direct effect of the gene-III lesion itself, and not polar effects on other genes. fCA55 Is abnormal in two respects. First, it is noninfective; in this it resembles another nonpolar gene-III deletion mutant, fKN16, which is missing 507 bp encompassing roughly the first half of the gene. Second, it is secreted as polyphage--very long particles containing many unit-length DNA molecules; in this respect, fCA55 differs from fKN16. When the viral proteins of these two mutants were analyzed with antibody directed against gene-III protein, it was found that fKN16 contains an altered gene-III protein, while fCA55 is unreactive. It was concluded that the gene-III protein has two functional domains: the N-terminal domain, missing in both mutants, is required for viral infectivity; while the C-terminal domain, partly missing in fCA55 but retained in fKN16, is incorporated into the virion, and is responsible for the protein's role in generating normal, unit-length particles.

Mycoplasma gateae arthritis and tenosynovitis in cats: case report and experimental reproduction of the disease.

Polyarthritis and tenosynovitis were diagnosed in a cat. Clinical signs of 2 months' duration included swollen limbs, painful joints (sensitive to touch), lameness, and pyrexia. Laboratory test data revealed hypogammaglobulinemia, hypoalbuminemia, leukocytosis, and mild anemia. The cat was euthanatized and necropsied; there were chronic necrotizing fibrinopurulent tenosynovitis and arthritis with bone and cartilage erosions. Cultural examinations of synovia were positive for Mycoplasma gateae, but bacterial and viral cultural examinations were negative. Organisms propagated from the M gateae isolate were inoculated IV into 6 specific-pathogen-free cats--3 of these being subjected to immunosuppression induced with azathioprine. The 6 inoculated cats became lame 5 to 9 days later, and 5 became febrile. Cultural examinations of the pharynx in 4 cats were positive for M gateae and in 3 cats, the organism was isolated from various joints. Microscopically, arthritis and tenosynovitis were identified in all cats. Two specific-pathogen-free cats were used as controls (noninoculated); these did not become lame, had negative M gateae cultures, and were free of histopathologic abnormalities. Reproduction of disease with recovery of the causative agent indicates the pathogenicity of this particular isolate of M gateae in the cat when inoculated IV.

Fibrinous pericarditis in the horse.

During a period of 18 months, between July 1978 and January 1980, 4 adult horses were referred to the New York State College of Veterinary Medicine with evidence of congestive heart failure. Characteristic clinical abnormalities included marked muffling of heart sounds, tachycardia, jugular vein distention, and peripheral edema. Treatment with antibiotics, diuretics, and anti-inflammatory drugs was unsuccessful, and all four died or were euthanatized and necropsied. At necropsy, there was marked distention of the pericardial sac with fluid, and thick layers of fibrin were deposited uniformly over the epicardium. In 3 cases, attempts to isolate bacteria and viruses from pericardial fluid were unsuccessful; in the 4th case, Actinobacillus equuli was isolated on culture of the pericardial fluid.

Bilateral lower palpebral demodicosis in a dairy cow.

A registered 3 year old Guernsey heifer, recently fresh, was examined for sudden appearance of diffuse, firm swelling beneath both eyes. Vital signs and physical examination were within normal limits; no lymphadenopathy was present. Biopsy revealed a chronic eosinophilic granulomatous cellulitis and degenerate Demodex bovis. the lesions selfresolved within three months.