Effects of PBBs on cattle. III. Target organ modification as shown by renal function and liver biochemistry.

Efforts were made to more clearly delineate target organs and mechanisms of toxicity for PBBs in cattle. Methods were developed to obtain sequential liver biopsies on bovine heifers which yield 0.5 to 1.0 g of tissue. PBB was fed at a dose of 250 mg/head/day to Holstein heifers for 202 days. This dose produced no clinical signs of toxicity in any of the heifers, yet this produced tissue PBB concentration of greater than 100 times the FDA tolerance in body fat of 0.3 ppm. Liver biopsies (0.5-1.0 g each) were taken at days 0, 90, and 180. The liver tissue was homogenized and microsomes were prepared. Dithionite difference spectra were determined on the carbon monoxide treated microsome suspension and the cytochrome P-450 content determined. Also, the 100,000g supernatant was saved for ornithine decarboxylase analysis as a measure of hepatocyte proliferative activity. Results of the cytochrome P-450 analysis showed a significant (p < 0.05) two-fold elevation (per gram of wet liver) by day 90 and remained significantly (p < 0.05) elevated on day 180. The cytochrome P-450 values of control animals not receiving PBBs showed no such increase with time. The biopsy procedure appeared not to adversely affect the liver cytochrome P-450 concentration in the control heifers. These results show that PBBs at a dose of 250 mg/day induced the drug metabolism system of the liver, of which the cytochrome P-450 is a part, indicating that the liver is a potential target organ for PBBs. However, this has not been shown to cause clear signs of hepatotoxicity in the cow as determined from histopathology or serum enzyme analyses. The observed elevation of gross liver weights of the PBB-treated animals might be an expected consequence of the cytochrome P-450 induction. In contrast to rodents, the kidney has been identified by histopathology as a target organ for PBB toxicity in cattle. However, renal function studies with (131)I-sodium-iodohippurate and (125)I-sodium iodothalamate in PBB treated cows indicated that PBB toxicity to the kidney did not affect glomerular filtration rate or effective renal plasma flow even though nephrotoxic effects were produced. From these studies, both liver (as expected) and kidney (unexpected) were affected by PBBs. For liver this did not result in hepatotoxicity while for kidney nephrotoxicity was produced but could not be mechanistically explained.

The alpha folate receptor is highly activated in malignant pleural mesothelioma.

OBJECTIVE: To determine whether the folate receptor gene is overexpressed in malignant pleural mesothelioma. METHODS: Differential display analysis was performed with fresh frozen RNA obtained from normal lung, pleura, and mesothelioma. Sixty differentially expressed genes were identified and characterized. One gene that was over-expressed in mesothelioma versus normal tissue was the human alpha folate receptor. In situ hybridization with antisense probes designed on the basis of the sequence of the folate receptor was performed with frozen sections from 61 patients (33 epithelial and 28 mixed or sarcomatoid tumors) with malignant pleural mesothelioma. The controls included normal pleura, normal lung, other cancers, and sense controls for all of the tumors. Northern analysis with a folate receptor probe and immunohistochemical analysis with anti-alpha folate receptor antibodies were also performed. RESULTS: Forty-four (72%) of the 61 mesothelioma tumors were found to have between 2-fold and 4-fold higher mRNA expression of the folate receptor when compared with the control tissues. The histologic type of the tumor did not affect the rate of folate receptor activation. Northern analysis and immunohistochemical experiments confirmed these findings. CONCLUSIONS: A majority of mesothelioma tumors examined overexpress the alpha folate receptor protein when compared with normal adjacent tissues. This finding may help explain the observations that antifolate drugs have activity in the treatment of mesothelioma. It also encourages further study of folate receptor-related treatment strategies in this malignancy.

Histologic changes in the seminiferous tubules after vasectomy.

PIP: 4 different studies on the effect of vasectomy on the testis in canines and humans are reported. Testicular biopsies were performed at certain intervals and the results in both humans and dogs were nearly identical. It was found that spermatogenesis 2 to 3 weeks after vasectomy remained unchanged with accumulation of spermatozoa in the tubules. Between 3 and 6 weeks, progressive spermatogenic arrest with few spermatozoa and decreased spermatids were observed. Between 100 and 300 days, occasional mature sperm were found in the tubules indicating a return of spermatogenesis. Meiotic studies showed this to be an arrest in early prophase. It is theorized that spermatogenesis may be sensitive to pressure changes in the tubular system.^ieng

Factors affecting serum oxytetracycline levels in beef calves.

Fifteen Aberdeen Angus steers, 295-364 kg, were dosed with either 4.4 or 11 mg of oxytetracycline hydrochloride/kg im. The antimicrobial activity of the serum was determined periodically, and the resulting data were treated statistically to determine the sources of variation. Variance in serum levels of oxytetracycline activity was attributed to dose, time of bleeding, order of dosing, animal, and assay. The total variance component was proportionately greater for the 11-mg/kg dose than for the 4.4-mg/kg dose. Animal variance increased with the higher dose level of oxytetracycline. The influence of dose on serum level was tested by applying a t test to the mean serum levels and their standard deviations at each bleeding time. The 4.4- and 11-mg/kg serum levels were significantly different (p less than 0.01) at all bleeding times. The 4.4-mg/kg serum levels mutliplied by 2.5 were not significantly different (p less than 0.05) from the 11-mg/kg serum levels at all bleeding times.

Stimulation of goblet cell mucous secretion by activation of nerves in rat conjunctiva.

An epithelial debridement wound, as a stimulus to the cornea, causes conjunctival goblet cell mucous secretion in that eye. To determine if this stimulation of secretion is neurally mediated, rats were anesthetized and the local anesthetic lidocaine (1%) or buffer alone was administered topically and/or subconjunctivally for 15 min. A corneal epithelial debridement wound was made in one eye. The contralateral eye served as the control. After 5-120 min, animals were sacrificed and inferior bulbar conjunctival buttons removed. Mucus in the goblet cells was stained with Alcian blue and periodic acid-Schiff's reagent to indicate mucin-containing goblet cells. The number of mucin-containing goblet cells/0.16 mm2 was determined by light microscopy; a decrease in number indicated an increase in mucous secretion. Stimulation by corneal wounding induced goblet cell mucous secretion in that eye. Secretion was observed as rapidly as 5 min after stimulus and for as long as 120 min. Topical application of lidocaine, subconjunctival injection of lidocaine, or a combination of both inhibited wound-induced stimulation of mucous secretion. We conclude that conjunctival goblet cell mucous secretion can be neurally mediated and could serve as an immediate response to protect the ocular surface.

Localization of nerves adjacent to goblet cells in rat conjunctiva.

Neural stimulation of the cornea induces conjunctival goblet cell mucous secretion. Immunofluorescence microscopy was used to determine if nerves are present near conjunctival goblet cells and what types of nerves are present. In euthanized rats, the local anesthetic lidocaine (1%) was placed topically on the ocular surface for 10 min to prevent goblet cell mucous secretion. The ocular surface tissues were removed and either fixed in formaldehyde and then frozen, or frozen first and then post-fixed in formaldehyde. Tissue was sectioned and nerves localized by indirect immunofluorescence microscopy, using antibodies to synaptophysin (indicates nerve, independent of type), vasoactive intestinal peptide (VIP, indicates parasympathetic nerves), tyrosine hydroxylase (TH, indicates sympathetic nerves), dopamine beta-hydroxylase (DBH, indicates sympathetic nerves), phenylethanolamine-N-methyltransferase (PNMT, indicates sympathetic nerves), and calcitonin gene-related peptide (CGRP, indicates sensory nerves). Goblet cells were identified by phase-contrast microscopy. Synpatophysin-containing nerves were present in the basolateral region of conjunctival goblet cells clusters. Nerve fibers immunoreactive to VIP were found in the conjunctiva along the epithelial-stromal junction and around the basolateral aspect of goblet cell clusters. Nerve fibers immunoreactive to TH and DBH were detected surrounding goblet cells and in the conjunctival stroma. Nerve fibers immunoreactive to CGRP were detected in the epithelium and at the epithelial stromal junction, but were not localized near goblet cell clusters. CGRP-containing nerve fibers were also detected in the conjunctival stroma under the epithelium. We conclude that efferent parasympathetic and sympathetic, but not afferent sensory, nerves appear to be located adjacent to conjunctival goblet cell clusters. Activation of efferent parasympathetic and sympathetic nerves could directly stimulate conjunctival goblet cell mucous secretion. Antidromic activation of afferent sensory nerves releasing neurotransmitters could stimulate goblet cell secretion by a paracrine mechanism.

Therapeutic responses of piglets to experimentally induced colibacillosis.

The purpose of these experiments was to determine the effects of prior exposure to antimicrobials on subsequent treatment of artificially induced colibacillosis in pigs. One- to two-week-old piglets were given 10(7) multiple antibiotic resistant Escherichia coli orally. Two groups of the piglets received priming doses at different levels of the antimicrobial preparation, ASP-250 before challenge. The remaining group received no antimicrobial. Piglets ill as a result of the challenge were treated with chloramphenicol of chlortetracycline, or received no treatment. Chloramphenicol was significantly more effective than chlortetracycline in terminating colibacillosis in the primed and unprimed groups. There were fewer deaths and relapse cases in those groups which received chloramphenicol treatment. Results in piglets treated with chlortetracycline were not significantly better than those which received no treatment.

Nonlactating-cow therapy with a formulation of penicillin and novobiocin: therapeutic and prophylactic effects.

An experimental product incorporating 500,000 IU of procaine penicillin G and 600 mg of sodium novobiocin in 2% aluminum monostearate-peanut oil gel (10-ml dose) was used to treat all quarters of 56 cows which were infected in at least 1 quarter at time of final mild-out at end of lactation. Treatment was withheld from 89 cows uninfected in all quarters. Quarter infection was determined by bacteriologic culturing of milk samples collected at the last regular milking, at intervals up to final milk-out (7 or 12 days later), at calving, and 1 week later. Clearance rates against Staphylococcus aureus, Staphylococcus epidermidis, streptococci other than Streptococcus agalactiae, and coliform bacteria in treated quarters were 83, 94, 88, and 71%, respectively. Subtraction of the spontaneous clearance rate of about 50% in untreated quarters resulted in values of 35 to 45% for drug efficacy against existing staphylococcal and streptococcal infections. Prophylactic efficacy was examined. In cows entering the true nonlactating period with 1 or more quarters infected, new infection rates across the period aming quarters uninfected at the beginning were 36.0% among untreated cows and 6.3% among treated cows (P less than 0.005). The comparable rates for cows entering the nonlactating period uninfected in all quarters were 5.7 and 0%. Staphylococcus aureus and streptococci, which comprised 38.5% of new period infections among untreated cows, were completely lacking among treated cows (P less than 0.025). Within the treated group of cows, 83.1% of infected quarters were cleared, and new infection rate in the non-lactating period was 50% less than the rate among untreated cows. Because the frequency of intramammary infection in this herd was quite low at "drying-off" (10.5% of quarters), the net effect on herd health of selective therapy of cows infected at end of lactation was a reduction in total quarter infection from 19.8 to 13.6%.

Nonlactating-cow therapy with a formulation of penicillin and novobiocin: mammary irritation and residues.

An experimental product incorporating 500,000 IU of procaine penicillin G and 600 mg of sodium novobiocin in 2% aluminum monostearate-peanut oil gel (10-ml dose) was infused after the final milk-out at end of lactation into all 4 mammary quarters of 56 cows that were infected in at least 1 quarter. The therapeutic and prophylactic efficacies were published in the companion report. Infusion of the product in all quarters of 5 lactating cows resulted in only slight irritation. Penicillin was eliminated by the 11th milking and novobiocin by the 5th. After infusion in the dry udder, the antibiotics were no longer detectable in serous secretion after 14 days and failed to appear in urine at the earliest (7-day) sampling after administration. Neither antibiotic was detectable in the 1st postpartum milking after nonlactating periods as short as 3 weeks.

Excretion of [3H]prednisolone in clinically normal and experimentally infected bovine udders.

The excretion rate of [3H]prednisolone from clinically normal and experimentally infected udders of 10 lactating cows was studied. Each quarter of 6 cows was injected with a single dose of [3H]prednisolone mixed with non-radioactive prednisolone equivalent to 10 mg in 10 ml of peanut oil base. Each of the remaining 4 cows was given 40 mg of nonradioactive prednisolone and [3H]prednisolone in 60% ethanol IV. Control and postadministration samples of blood, milk, and urine were examined for radioactivity. The effects of [3H]prednisolone were evaluated in the same cows, first in clinically normal udders, then 2 weeks later in udders experimentally infected with Streptococcus agalactiae. Absorption and elimination of prednisolone were the same before and after induced infection. Within 3 hours after intramammary injection, 95% of the labeled prednisolone was absorbed systemically, less than 5% of this dose was recovered in milk, and 29% was excreted in urine. After IV injection of [3H]prednisolone, less than 0.2% of the total radioactivity was recovered in milk and less than 46% was excreted in urine. Clinical mastitis induced by S agalactiae was moderate. Circulating blood leukocytes and somatic cells in the milk of normal cows remained essentially unchanged. The leukocyte response to induced infection was rapid in blood and milk. Large numbers of leukocytes were noticed in the milk and a severe leukopenia occurred. Prednisolone treatment did not alter the number of somatic cells in milk or reduce the inflammatory response of experimentally infected cows.

Use of the double-isotope, single-injection method for estimating renal function in normal and polybrominated biphenyl-exposed dairy cows.

A method is described for conducting a rapid and efficient renal function test in dairy cattle. The method, adapted from methods used for man and dogs, utilizes radiolabeled 131I-sodium iodohippurate to determine effective renal plasma flow (ERPF) and 125I-sodium iothalamate to determine glomerular filtration rate (GFR). The mean GFR for adult cattle was determined to be 2.8 +/- 0.4 ml/kg/minute with a biological half-life of 35.5 +/- 1.4 minutes. The mean ERPF was found to be 10.5 +/- 1.9 ml/kg/minute with a half-life of 17.9 +/- 0.6 minutes. These values are comparable with those in man, but are lower than values in dogs. A toxcity study was done with dairy cattle exposed to polybrominated biphenyls (PBB). Efforts were made to determine the amount of time required for kidney lesions to develop and, if possible, to delineate the potential site of action of PBB. Apparently PBB do not affect GFR or ERPF, even though they produce nephrotoxic effects. Potential mechanisms to explain these results are described.

Use of the double-isotope single-injection method for estimating renal function in normal cross-bred swine.

The double-isotope single-injection method to estimate renal function which utilizes the radiolabeled chemicals [131I]sodium iodohippurate and [125I]sodium iothalamate was evaluated in swine. A total of 46 normal, cross-bred swine were used to determine the applicability of this method for measuring the glomerular filtration rate and effective renal plasma flow. The mean glomerular filtration rate in pigs was determined to be 5.33 +/- 0.82 ml/kg of body weight/minute for [125I]sodium iothalamate with a biological half-life (T 1/2) of 39.18 +/- 7.44 minutes. The mean effective renal plasma flow was determined to be 19.25 +/- 3.12 ml/kg of body weight/minute for [313I]sodium iodohippurate, with a T 1/2 of 18.45 +/- 1.74 minutes. These values are more closely related to the glomerular filtration and effective renal plasma flow values reported for dogs and cats than they are to values reported for man. The method is rapid and reliable; results are available 6 to 8 hours after the experiment. This method is advantageous when information about renal function variables is a prerequisite to pharmacokinetic or toxicologic studies.

Pathology of experimentally induced polybrominated biphenyl toxicosis in pregnant heifers.

Toxicosis was induced in pregnant Holstein-Friesian heifers by giving polybrominated biphenyls a in gelatin capsules at the rate of 25 g/day. Initially, this dosage was approximately 67 mg/kg of body weight. Clinical signs were anorexia, excessive lacrimation and salivation, diarrhea, emaciation, dehydration, depression, and abortion. Fever was not evident during the experiment. Values for serum glutamic-oxalacetic transaminase, lactic dehydrogenase, blood urea nitrogen, and bilirubin were increased. Changes in packed cell volume, hemoglobin content, total erythrocyte and leukocyte counts, and differential leukocyte counts were minimal and reflected dehydration and secondary infection. The principal urine changes were decreased specific gravity and moderate proteinuria. Gross necropsy findings included dehydration; subcutaneous emphysema and hemorrhage; atrophy of the thymus; fetal death with concomitant necrosis of cotyledons; kidneys that were enlarged, pale tan to gray; thickened wall of the gallbladder; inspissated bile; edema of abomasal folds; mucoid enteritis; linear hemorrhage and edema of the rectal mucosa; and secondary pneumonia. Microscopic changes were most marked in the kidneys, gallbladder, and eyelid. In the kidney, the principal changes were extreme dilatation of collecting ducts and convoluted tubules, with epithelial degenerative changes of cloudy swelling, hydropic degeneration, and separation from the basement membrane. Common changes in the gallbladder were moderate to marked hyperplasia and cystic dilatation of the mucous glands in the lamina propria. The changes in the eyelids were characterized by hyperkeratosis, with accumulations of keratin in hair follicles of the epidermis and squamous metaplasia with keratin cysts in the tarsal glands. Clinical signs and lesions of toxicosis did not develop in heifers given the polybrominated biphenyls at the rate of 0.25 mg and 250 mg/day for 60 days. Initially these rates were approximately 0.00065 mg/kg and 0.65 mg/kg of body weight, respectively.

Undergraduate orthodontics--a collision course on occlusion.

Five years ago the dental school at Belfast instituted a 'longitudinal course' in orthodontics. It has meant changes to attitudes and working practices, and staff and students have been both flexible and open minded. Here, Andrew Richardson and Hill Mercer, outline the course, the contortions, and some of the problems that have been straightened out.