Home-based vs inpatient education for children newly diagnosed with type 1 diabetes.

BACKGROUND: Initial management of children diagnosed with type 1 diabetes (T1D) varies worldwide with sparse high quality evidence regarding the impact of different models of care. AIM: To compare the inpatient model of care with a hybrid home-based alternative, examining metabolic and psychosocial outcomes, diabetes knowledge, length of stay, and patient satisfaction. SUBJECTS AND METHODS: The study design was a randomized-controlled trial. Inclusion criteria were: newly diagnosed T1D, aged 3 to 16 years, living within approximately 1 hour of the hospital, English-speaking, access to transport, absence of significant medical or psychosocial comorbidity. Patients were randomized to standard care with a 5 to 6 day initial inpatient stay or discharge after 2 days for home-based management. All patients received practical skills training in the first 48 hours. The intervention group was visited twice/day by a nurse for 2 days to assist with injections, then a multi-disciplinary team made 3 home visits over 2 weeks to complete education. Patients were followed up for 12 months. Clinical outcomes included HbA1c, hypoglycemia, and diabetes-related readmissions. Surveys measured patient satisfaction, diabetes knowledge, family impact, and quality of life. RESULTS: Fifty patients were recruited, 25 to each group. There were no differences in medical or psychosocial outcomes or diabetes knowledge. Average length of admission was 1.9 days shorter for the intervention group. Families indicated that with hindsight, most would choose home- over hospital-based management. CONCLUSIONS: With adequate support, children newly diagnosed with T1D can be safely managed at home following practical skills training.

Larger numbers of CD4(bright) dendritic cells in donor bone marrow are associated with increased relapse after allogeneic bone marrow transplantation.

Relapse is the major cause of death after allogeneic bone marrow transplantation (BMT). This study tested the hypothesis that the numbers of donor mononuclear cells, lymphocytes, and CD34(+) cells influence relapse and event-free survival (EFS) after BMT. The study population consisted of 113 consecutive patients with hematologic malignancies who underwent non-T-cell-depleted BMT from HLA-matched siblings. Sixty-four patients had low-risk diagnoses (ALL/AML CR1, MDS RA/RARS, and CML CP1); 49 patients had high-risk diagnoses (all others). CD34(+) cells, T cells, B cells, natural killer cells, monocytes, and a rare population of CD3(-), CD4(bright) cells in the allografts were measured by flow cytometry. The CD3(-), CD4(bright) cells in bone marrow had the same frequency and phenotype as CD123(bright) type 2 dendritic cell (DC) progenitors, and they differentiated into typical DCs after short-term culture. Cox regression analyses evaluated risk strata, age, gender, and the numbers of nucleated cells, CD3(+) T cells, CD34(+) hematopoietic cells, and CD4(bright) cells as covariates for EFS, relapse, and nonrelapse mortality. Recipients of larger numbers of CD4(bright) cells had significantly lower EFS, a lower incidence of chronic graft-versus-host disease (cGVHD), and an increased incidence of relapse. Recipients of larger numbers of CD34(+) cells had improved EFS; recipients of fewer CD34(+) cells had delayed hematopoietic engraftment and increased death from infections. In conclusion, the content of donor CD4(bright) cells was associated with decreased cGVHD and graft-versus-leukemia effects in recipients of allogeneic bone marrow transplantation, consistent with a role for donor DCs in determining immune responses after allogeneic BMT.

High dose chemotherapy without hematopoietic cell support for the treatment of refractory lymphoma.

Conventional dose combination chemotherapy for patients with relapsed or refractory lymphoma is rarely curative. High dose chemotherapy followed by hematopoietic progenitor cell transplant (HPCT) has a clearly defined role in patients who have first relapsed after standard CHOP chemotherapy for lymphoma. However, the role of HPCT is less well defined for patients with chemo-resistant, or chemo-refractory disease. Sixteen patients (15 Non-Hodgkin's, 1 Hodgkin's Disease) were entered into a phase II study to determine if a dose intensive induction regimen in heavily pre-treated refractory lymphoma patients could permit further consolidation with HPCT. The primary endpoints were survival, response, toxicity, and resource utilization. The regimen consisted of continuous infusion etoposide 1 or 2 gm/m2/72 hours, idarubicin 12 mg/m2/d for 3 days followed by cytarabine 2 gm/m2/72 hours on days 8, 9, and 10 (VIC). Fifteen patients were evaluable for objective response. The overall response rate was 53% with 7 patients achieving a partial response and 1 patient achieving a complete response. Of the 8 responders, 6 patients subsequently received high dose chemotherapy followed by HPCT (4 autologous, 2 allogeneic). The median survival was 176 days for the non-responders contrasted with 722 days for the responders. The average duration of hospitalization was 38 days. Toxicity was mainfest primarily as mucositis with a median grade of 3 among the first 13 patients, and a median grade of 2 in three subsequent patients who received an etoposide dose of 1 gm/m2/72 hours. All patients had an episode of neutropenic fever and 5 patients developed clinically significant pneumonitis during therapy. The VIC regimen is active in the treatment of chemo-refractory lymphoma with clinically significant differences in survival for patients who respond to therapy. Further modifications to the regimen could include the addition of a topoisomerase I inhibitor for synergy with etoposide, and using VIC as part of a tandem transplant regimen where response to VIC would allow further therapy with a myeloablative induction followed by HPCT.

Immunochemical analysis of quinol-thioether-derived covalent protein adducts in rodent species sensitive and resistant to quinol-thioether-mediated nephrotoxicity.

2,3,5-Tris(glutathion-S-yl)hydroquinone (TGHQ) is nephrotoxic in male Fischer 344 rats (20 micromol/kg) and albino guinea pigs (200 micromol/kg), but not BALB/c or B6C3F1 mice or Golden Syrian hamsters (200 micromol/kg). Since quinones are known to alkylate proteins, and because such macromolecular damage may play a role in cytotoxicity, we investigated the covalent binding of TGHQ to kidney (target tissue) and liver (nontarget tissue) of rodents "sensitive" or "resistant" to the nephrotoxic effects of TGHQ. Immunohistochemical staining of tissue obtained 2 h after administration of TGHQ, with rabbit anti-2-bromo-N-(acetyl-L-cystein-S-yl)HQ antibodies, correlated with the subsequent region of necrosis observed 19 h after dosing in Fischer 344 rats and guinea pigs. Immunohistochemical staining was localized to the S3 segment of the renal proximal tubules, at the corticomedullary junction along the medullary rays, and in the outer stripe of the outer medulla. Immunostaining was also observed in the same region in hamsters, but subsequent necrosis did not develop. In contrast, no immunostaining was observed in mice. Moreover, immunostaining was not detected in the livers of any species. Western blot analysis revealed numerous immunoreactive renal proteins in TGHQ-treated animals. The most distinctive immunostaining renal proteins were observed in Fischer 344 rats at approximately 34 kDa (mitochondria), approximately 35 kDa (nuclei) which comigrated with histone H1, and approximately 73 kDa (urine) which comigrated with gamma-glutamyl transpeptidase. These adducted proteins were not detected in other species. Qualitative differences in alkylated proteins may therefore contribute to species susceptibility to TGHQ.

Progression of borderline increases in albuminuria in adolescents with insulin-dependent diabetes mellitus.

We aimed to determine the natural history of borderline increases in albuminuria in adolescents with insulin-dependent (Type 1) diabetes mellitus (IDDM) and factors which are associated with progression to persistent microalbuminura. Fifty-five normotensive adolescents with IDDM and intermittent microalbuminura (overnight albumin excretion ratte of 20-200 micrograms min-1 on one of three consecutive timed collections, n = 29) or borderline albuminura (mean overnight albumin excretion rate of 7.2-20 micrograms min-1 on one of three consecutive timed collections, n = 30) were followed prospectively at 3 monthly intervals. The endpoint was persistent microalbuminuria defined as a minimum of three of four consecutive overnight albumin excretion rates of greater than 20 micrograms min-1. One hundred and forty-two adolescents with IDDM and normoalbuminura were also followed prospectively. Fifteen of the 59 patients (25.4%) with intermittent (9/29) or borderline (6/30) albuminura progressed to persistent microalbuminura (progressors) over 28 (15-50) months [median (range)] in comparison with two of the 142 patients with normoalbuminuria at entry (relative risk = 12.6; p = 0.001). Progressors to persistent microalbuminura were pubertal and had higher systolic (p = 0.02) and diastolic (p = 0.02) blood pressure, and HbA1c (p = 0.004) than non-progressors. All patients remained normotensive. Glomerular filtration rate, apolipoproteins, dietary phosphorus, protein and sodium intakes, and prevalence of smoking did not differ between progressors and non-progressors. Total renin was higher in the diabetic patients without a difference between progressors and non-progressors. In conclusion there is a relatively high rate of progression to persistent microalbuminuria in pubertal adolescents with borderline increases in albuminura and duration greater than 3 years. These patients require attention to minimize associated factors of poor metabolic control and higher blood pressure in the development of incipient nephropathy.

Cytotoxicity and cell-proliferation induced by the nephrocarcinogen hydroquinone and its nephrotoxic metabolite 2,3,5-(tris-glutathion-S-yl)hydroquinone.

Hydroquinone, an intermediate used in the chemical industry and a metabolite of benzene, is a nephrocarcinogen in the 2-year National Toxicology Program bioassay in male Fischer 344 rats. Current evidence suggests that certain chemicals may induce carcinogenesis by a mechanism involving cytotoxicity, followed by sustained regenerative hyperplasia and ultimately tumor formation. Glutathione (GSH) conjugates of a variety of hydroquinones are potent nephrotoxicants, and we now report on the effect of hydroquinone and 2,3,5-(tris-glutathion-S-yl)hydroquinone, on site-selective cytotoxicity and cell proliferation in rat kidney. Male Fischer 344 rats (160-200 g) were treated with hydroquinone (1.8 mmol/kg or 4.5 mmol/kg, p.o.) or 2,3,5-(tris-glutathion-S-yl)hydroquinone (7.5 micromol/kg; 1.2-1.5 micromol/rat, i.v.), and blood urea nitrogen (BUN), urinary gamma-glutamyl transpeptidase (gamma-GT), alkaline phosphatase (ALP), glutathione-S-transferase (GST) and glucose were measured as indices of nephrotoxicity. Hydroquinone (1.8 mmol/kg, p.o.) is nephrotoxic in some rats, but not others, but cell proliferation (BrDU incorporation) in proximal tubular cells of the S3M region correlates with the degree of toxicity in individual rats. At 4.5 mmol/kg, hydroquinone causes significant increases in the urinary excretion of gamma-GT, ALP and GST. Pretreatment of rats with acivicin prevents hydroquinone-mediated nephrotoxicity, indicating that toxicity is dependent on the formation of metabolites that require processing by gamma-GT. Consistent with this view, 2,3,5-(tris-glutathion-S-yl)hydroquinone, a metabolite of hydroquinone, causes increases in BUN, urinary gamma-GT and ALP, all of which are maximal 12 h after administration of 2,3,5-(tris-glutathion-S-yl)hydroquinone. In contrast, the maximal excretion of GST and glucose occurs after 24 h. By 72 h, BUN and glucose concentrations return to control levels, while gamma-GT, ALP and GST remain slightly elevated. Examination of kidney slices by light microscopy revealed the presence of tubular necrosis in the S3M segment of the proximal tubule, extending into the medullary rays. Cell proliferation rates in this region were 2.4, 6.9, 15.3 and 14.3% after 12, 24, 48 and 72 h, respectively, compared to 0.8-2.4% in vehicle controls. Together with the metabolic data, the results indicate a role for hydroquinone-thioether metabolites in hydroquinone toxicity and carcinogenicity.

Induction of a permeability transition in rat kidney mitochondria by pentachlorobutadienyl cysteine: a beta-lyase-independent process.

A Ca2+-dependent inner mitochondrial membrane permeability transition is induced by a number of agents, an effect which is thought to cause cytotoxicity. This transition involves formation of a pore allowing the passage of solutes of up to 1500 Da; it is blocked by cyclosporine A and Ca2+ chelating agents. The mitochondrial nephrotoxicant S-(1,2,3,4, 4-pentachlorobutadienyl)-L-cysteine (PCBC) caused collapse of the mitochondrial membrane potential, Ca2+-independent oxidation of pyridine nucleotides and release of accumulated Ca2+ in isolated rat kidney mitochondria, three hallmarks of the permeability transition. These effects were blocked by cyclosporine A and by ethylene glycol bis(beta-aminoethyl ether) tetraacetic acid (EGTA). Furthermore, EGTA was capable of reversing the collapse of the membrane potential. These data indicate that PCBC induced an inner membrane permeability transition. Interestingly, addition of aminoxyacetic acid, a beta-lyase inhibitor, did not prevent the permeability transition, and a nonmetabolizable analog of PCBC, S-(1,2,3,4, 4-pentachlorobutadienyl)-L-alpha-methyl cysteine, induced the permeability transition. Thus PCBC may act to induce the permeability transition through a mechanism that does not require metabolism by a beta-lyase. Since metabolism by a beta-lyase is required for PCBC toxicity, it is not clear that the permeability transition is involved in cysteine conjugate-mediated renal cell injury.

Glutathione conjugates of tert-butyl-hydroquinone, a metabolite of the urinary tract tumor promoter 3-tert-butyl-hydroxyanisole, are toxic to kidney and bladder.

3-tert-Butyl-4-hydroxyanisole and tert-butyl-hydroquinone (TBHQ) are antioxidants known to promote renal and bladder carcinogenesis in the rat, although the mechanisms of these effects are unclear. Because glutathione (GSH) conjugates of a variety of hydroquinones are nephrotoxic, and because 2-tert-butyl-5-(glutathion-S-yl)hydroquinone [5-(GSyl)TBHQ], 2-tert-butyl-6-(glutathion-S-yl)hydroquinone [6-(GSyl)TBHQ], and 2-tert-butyl-3,6-bis-(glutathion-S-yl)hydroquinone [3,6-bis-(GSyl)-TBHQ] have been identified recently as metabolites of TBHQ in the male rat, we investigated the effects of these metabolites in the male rat. At the highest dose tested (400 micromol/kg,i.v.) 5-(Gsyl)TBHQ and 6-(GSyl)TBHQ caused 2-fold increases in the urinary excretion of gamma-glutamyl transpeptidase and alkaline phosphatase, and pigments arising from the polymerization of metabolites were deposited in the kidney. 3,6-bis-(GSyl)TBHQ (200 micromol/kg) was the most potent of the GSH conjugates tested and produced significant increases in the urinary excretion of gamma-glutamyl transpeptidase, alkaline phosphatase, lactate dehydrogenase, and glucose (2-, 2-, 22-, and 11-fold increases, respectively). Alterations in the biochemical parameters correlated with the degree of single cell and tubular necrosis in the S(3)-M segment of the proximal tubule, as observed by light microscopy. In addition to nephrotoxicity, 3,6-bis-(GSyl)TBHQ increased the bladder wet weight 2-fold and caused severe hemorrhaging of the bladder. The half-wave oxidation potentials of 5-(Gsyl)TBHQ and 6-(GSyl)TBHQ were similar to that of TBHQ, whereas the half-wave oxidation potential of 3,6-bis-(Gsyl)TBHQ was approximately 100 mV higher than that of TBHQ. The TBHQ-GSH conjugates also catalyzed the formation of 8- hydroxydeoxyguanosine, indicating that GSH conjugation does not impair the redox activity of TBHQ. Because some chemicals may induce carcinogenesis by a mechanism involving cytotoxicity followed by sustained regenerative hyperplasia, our results suggest that the toxicity of GSH conjugates of TBHQ to kidney and bladder may contribute to the promoting effect of 3-tert-butyl-4-hydroxyanisole and TBHQ in these tissues.

Growth and growth hormone secretion after treatment for acute lymphoblastic leukemia in childhood. 18-Gy versus 24-Gy cranial irradiation.

PURPOSE: Cranial irradiation (CI) given during the first phase of treatment of childhood acute lymphoblastic leukemia (ALL) has been associated with significant long-term morbidity. As a result, the dose of radiotherapy has been reduced from 24 to 18 Gy to reduce the severity of these late effects. To compare the effects of 24 and 18 Gy CI on growth, puberty, and growth hormone (GH) secretion, a cohort of survivors of childhood ALL were studied. PATIENTS AND METHODS: Of a total of 48 children, 28 (14 boys, 14 girls) had received 24 Gy and 20 (eight boys, 12 girls) had received 18 Gy. Similar chemotherapy regimens had been used in both groups, and age at diagnosis (5.2 +/- 2.7 vs. 5.1 +/- 2.8 years, 18 Gy vs. 24 Gy) and mean height at diagnosis [standard deviation score (SDS) 0.17 +/- 0.17 vs. 0.05 +/- 0.17, 18 Gy vs. 24 Gy] were comparable. RESULTS: Growth rates in both groups did not differ for the first 5 years after diagnosis. After this time, however, a significant height decrease was observed in children who had received 24 Gy but not in children who had received 18 Gy (at 8 years the change in SDS from diagnosis was -0.32 +/- 0.14 vs. -0.73 +/- 0.16, 18 Gy vs. 24 Gy, p < 0.05). Menarche occurred earlier in the girls in the 24-Gy group (at 12.9 +/- 0.3 vs. 11.7 +/- 0.4 years of age, 18 Gy vs. 24 Gy, p < 0.02). Overnight GH concentrations (12-h sampling every 20 min) were reduced in both groups compared with healthy age-matched control children but were even lower in the 24-Gy group (12.7 +/- 0.7 mU/L vs. 7.9 +/- 0.6 vs. 6.1 +/- 0.5 [6.4 +/- 0.4 ng/ml vs. 3.9-0.3 vs. 3.1 +/- 0.3]; control vs. 18 Gy and 24 Gy, p < 0.001; 18 Gy vs. 24 Gy, p < 0.025). CONCLUSIONS: Although both doses of CI impair GH secretion, 24 Gy has a greater impact on growth in the long term. This effect may be exaggerated by the induction of early puberty in some children.

Early cellular events couple covalent binding of reactive metabolites to cell killing by nephrotoxic cysteine conjugates.

Addition of the nephrotoxic cysteine conjugate, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), to the LLC-PK1 line of renal epithelial cells leads to covalent binding of reactive intermediates followed by thiol depletion, lipid peroxidation, and cell death (Chen et al., 1990, J. Biol. Chem., 265:21603-21611). The present study was designed to determine if increased intracellular free calcium might play a role in this pathway of DCVC-induced toxicity by comparing the temporal relationships among increased intracellular free calcium, lipid peroxidation, and cytotoxicity. Intracellular free calcium increased 1 hr after DCVC treatment, long before LDH release occurred. The elevation of intracellular free calcium and cytotoxicity was prevented by inhibiting DCVC metabolism with AOA. The cell-permeable chelators, Quin-2AM and EGTA-AM, prevented the toxicity. Pretreatment of cells with a nontoxic concentration of ionomycin increased intracellular free calcium and potentiated DCVC-induced LDH release. However, the antioxidant, DPPD, which blocks lipid peroxidation and toxicity, did not affect the increase in intracellular free calcium, whereas buffering intracellular calcium with Quin-2AM or EGTA-AM blocked both lipid peroxidation and toxicity without preventing the depletion of nonprotein sulfhydryls by DCVC. Ruthenium red, an inhibitor of mitochondrial calcium uptake, also blocked cell death. We hypothesize that covalent binding of the reactive fragment from DCVC metabolism leads to deregulation of intracellular calcium homeostasis and elevation of intracellular free calcium. Increased intracellular free calcium may in turn be coupled to mitochondrial damage and the accumulation of endogenous oxidants which cause lipid peroxidation and cell death.

Early morphological and biochemical changes during 2-Br-(diglutathion-S-yl)hydroquinone-induced nephrotoxicity.

Early subcellular targets of 2-Br-(diglutathion-S-yl)hydroquinone (2-Br-(diGSyl)HQ)-mediated nephrotoxicity were investigated by morphological and biochemical criteria. After treatment of male Fischer 344 rats with 2-Br-(diGSyl)HQ (30 mumol/kg), proximal tubular morphology was examined by electron microscopy. Changes in the plasma membrane, nuclei, and endoplasmic reticulum were observed within 30 min of 2-Br-(diGSyl)HQ administration. These changes consisted of loss of the brush border membrane, margination of heterochromatin, and reorganization of the endoplasmic reticulum into discrete aggregates. The desquamation of the brush border membrane into the tubular lumen corresponded with the rapid excretion of gamma-glutamyl transpeptidase and alkaline phosphatase in urine. As the injury developed, cell swelling with loss of cytosolic density and loss of chromatin staining was observed, and between 2 and 4 hr the nuclei underwent extensive karyorrhexis and karyolysis. Agarose gel electrophoresis of DNA isolated from the corticomedullary junction at 4 hr exhibited extensive fragmentation, which was random in nature. Mitochondria assumed a condensed configuration 2 hr after 2-Br-(diGSyl)HQ administration, but this was not followed by high-amplitude swelling prior to cell death and necrosis. Biochemical assessment of mitochondria, isolated from 2-Br-(diGSyl)HQ-treated rats at 2 hr, exhibited a significant (20%) decrease in respiratory control ratios (RCR), a consequence of an increase in State 4 respiration. At later time points (8 hr) State 4 respiration returned to control values, but the respiratory control ratio (RCR) remained significantly depressed due to decreases in State 3 respiration. At this time blood urea nitrogen concentrations were significantly elevated (41 +/- 3, mean +/- SD, n = 10). The data suggest that the plasma membrane and the nucleus are early targets of 2-Br-(diGSyl)HQ-induced cytotoxicity, and that alterations in mitochondrial structure and respiratory function occur following the initial injury.

The effect of Trypanosoma evansi infection on the oestrous cycle of Friesian Holstein heifers.

The effect of Trypanosoma evansi infection on oestrous cycling was studied in 12 Friesian Holstein heifers. In the Phase 1 of the investigation, six heifers were infected with T. evansi, the remaining six acted as uninfected controls. Daily body temperature, packed red cell volume (PCV) and parasitaemia measurements were obtained from each animal for 90 days. The animals were examined for external signs of oestrous activity twice daily, blood samples were taken three times a week and subjected to an enzyme-linked immunosorbent assay to detect plasma progesterone. Body weights were measured weekly. Parasites were eliminated by trypanocidal drug treatment 90 days after infection. In Phase 2 of the trial, the uninfected heifers were injected with a different stock of parasites and monitoring was continued as before. Infection with T. evansi resulted in a marked reduction in the rate of weight gain, an increase in body temperatures and a fall in PCV values. Eleven of the heifers continued to cycle normally for the duration of the study, irrespective of their infective status. One animal which stopped cycling lost 16.2% of its pre-infection body weight as a result of the infection and cessation of oestrous activity was considered to have been due to weight loss.

Early events following challenge of rabbits with Trypanosoma evansi and T. evansi components.

The intradermal injection of Trypanosoma evansi or T. evansi components into rabbits evoked trypanosome-specific responses in the skin. The strongest responses, which were those against the parasite surface-associated components, had the characteristics of an immediate type hypersensitivity reaction, followed by a delayed type. The responses were greater in rabbits from which infections had been cleared by chemotherapy than in animals with patient infections. These findings suggest that variant surface glycoprotein (VSG)-specific antibody activity and immunosuppression are effective in the skin and influence the outcome of infection with T. evansi in previously infected animals.

Mechanism of the nephrogenic repair response. Studies on proliferation and vimentin expression after 35S-1,2-dichlorovinyl-L-cysteine nephrotoxicity in vivo and in cultured proximal tubule epithelial cells.

Studies were performed in vivo using 35S-(1,2-dichlorovinyl)-L-cysteine, a nephrotoxin that damages the S3 segment of the proximal tubule after metabolism to a reactive intermediate. Initiation of damage (35S covalent binding) was complete by 6 hour, and an early proliferative response was observed by 24 hour in the S2 or S3C segments. Necrosis in the S3M and increased blood urea nitrogen were maximal at 48 hours and were accompanied by an increase in proliferation of cells at the wound site. Regeneration was marked by the appearance of vimentin expressing cells that lacked brush border enzymes. The loss of differentiated character in the regenerative epithelium persisted after the proliferation (bromodeoxyuridine incorporation) had stopped; redifferentiation occurred between days 5 and 13. Much of the process was reproduced by culturing rat kidney proximal tubule epithelial cells in defined medium. As growth increased, the cells expressed vimentin and lost brush border marker enzymes. However, as the cells reached high density and stopped dividing there was an increase in brush border markers, as was seen in vivo. Vimentin expression did not decrease, however. The data support a mechanism for damage and nephrogenic repair composed of 1) interaction of the toxin with the target cells, 2) necrosis and exfoliation, 3) loss of differentiation and cell growth, 4) recovery of the damaged area and cessation of cell growth, and 5) differentiation of the quiescent cells. Nephrogenic repair may have similarities with the differentiation of the tubular epithelium during development.

Stimulation of N-methyl-D-aspartate receptor-mediated calcium entry into dissociated neurons by reduced and oxidized glutathione.

The effects of GSH (gamma-glutamylcysteinylglycine) and GSSG on intracellular calcium levels ([Ca2+]i) were investigated using fura-2-loaded dissociated brain cells from newborn rat pups. Both produced concentration-dependent increases in [Ca2+]i (EC50 values of 914.3 +/- 190.5 and 583.0 +/- 97.2 microM for GSH and GSSG, respectively), similar to that observed with N-methyl-D-aspartate (NMDA) and other agonists at the NMDA receptor. Maximum response (expressed as percentage change in [Ca2+]i relative to basal) was significantly greater for GSSG (37.5 +/- 1.6%) than for GSH (25.3 +/- 1.6%). The response to both agents was prevented or reversed by competitive (100 microM) (-)-2-amino-5- phosphonovalerate and noncompetitive (400 nM) MK-801 or 1.0 mM Mg2+ antagonists of NMDA receptor-mediated calcium entry, even at concentrations of GSH and GSSG normally producing maximal response. The idea that these effects are mediated, at least in part, by interaction with the NMDA receptor was supported by the effects of GSH and GSSG on the binding of the NMDA receptor ligand [3H]CGP-39653 to membranes isolated from hippocampal and cortical homogenates. Both GSH and GSSG displaced bound [3H]CGP-39653, with IC50 values of 0.93 +/- 0.18 and 11.02 +/- 1.22 microM, respectively, and produced an increase in the apparent Kd of binding (control, 8.92 +/- 0.83 nM, and GSH, 13.31 +/- 1.19 nM; control, 11.59 +/- 0.35 nM, and GSSG, 18.73 +/- 0.66 nM). However, both also produced modest reductions in Bmax (control, 1265 +/- 69 fmol/mg of protein, and GSH, 901 +/- 73 fmol/mg of protein; control, 1068 +/- 30 fmol/mg of protein, and GSSG, 730 +/- 18 fmol/mg of protein) and Hill slopes (GSH, 0.66 +/- 0.02; GSSG, 0.62 +/- 0.04). This suggests complex kinetics for the interaction of GSH and GSSG with the NMDA receptor. Taken together, the results suggest the potential for modulation of the NMDA receptor complex by GSH and GSSG.

Nephrotoxicity of 2-bromo-(cystein-S-yl) hydroquinone and 2-bromo-(N-acetyl-L-cystein-S-yl) hydroquinone thioethers.

The in vivo toxicity of isomeric cystein-S-yl and N-acetylcystein-S-yl conjugates of 2-bromohydroquinone was determined in male Sprague-Dawley rats. 2-Bromo-(dicystein-S-yl)hydroquinone [2-Br-(diCYS)HQ] and 2-bromo-(di-N-acetyl-L-cystein-S-yl)hydroquinone [2-Br-(diNAC)HQ] were considerably more nephrotoxic than their corresponding monosubstituted thioethers and 2-Br-(diCYS)HQ was more nephrotoxic than 2-Br-(diNAC)HQ. 2-Br-(diCYS)HQ caused elevations in blood urea nitrogen (BUN) concentrations and increases in the urinary excretion of glucose, lactate dehydrogenase (LDH), and gamma-glutamyl transpeptidase (gamma-GT) at a dose of 25 mumol/kg (iv). In contrast, 2-Br-(diNAC)HQ caused significant elevations in BUN at 100 mumol/kg and glucosuria and enzymuria at 50 mumol/kg. 2-Br-3-(CYS)HQ and 2-Br-5&6-(CYS)HQ caused increases in the biochemical indices of nephrotoxicity at doses between 50 and 150 mumol/kg whereas 2-Br-5-(NAC)HQ and 2-Br-6-(NAC)HQ required doses of 150-200 mumol/kg to cause smaller, though significant increases in urinary glucose, gamma-GT, and LDH excretion. The histological alterations caused by each thioether were qualitatively similar; only differences in the extent of the renal proximal tubular damage were observed. The initial lesion appears to involve the cells of the medullary ray and the S3M within the outer stripe of the outer medulla. The in vivo nephrotoxicity of 2-Br-(DiCYS)HQ, 2-Br-(diNAC)HQ, and the most potent monosubstituted thioethers, 2-Br-5&6-(CYS)HQ and 2-Br-6-(NAC)HQ, was investigated further. Pretreatment of animals with aminooxyacetic acid, an inhibitor of cysteine conjugate beta-lyase (beta-lyase), had no effect on the toxicity of 2-Br-(diCYS)HQ, partially inhibited the toxicity of 2-Br-5&6-(CYS)HQ, and almost completely protected against the toxicity of both 2-Br-6-(NAC)HQ and 2-Br-(diNAC)HQ. Thus, the nephrotoxicity of 2-Br-5&6-(CYS)HQ, 2-Br-6-(NAC)HQ, and 2-Br-(diNAC)HQ may be mediated, in part, via their processing by beta-lyase. Pretreatment of animals with probenecid, an inhibitor of renal organic anion transport, completely protected against the toxicity of 2-Br-(diNAC)HQ but had no effect on the toxicity of the other thioethers.

Some viral and protozool diseases in the European wildcat (Felis silvestris).

Ten European wildcats (Felis silvestris) were examined at necropsy and an additional 23 were examined clinically for evidence of viral diseases in Scotland. Two plasma samples taken from live free-living wildcats showed positive ELISA reactions to feline leukemia antigen. A feline leukemia virus of subgroup A was isolated from one of these samples, taken from a wildcat in north-western Scotland. No antibodies to feline coronavirus or feline immunodeficiency virus were detected in any sample. Three of the live wildcats and one of the dead had chronic mucopurulent rhinotracheitis suggestive of "cat flu." One other dead wildcat had diffuse enlargement of anterior lymph nodes. The findings indicated that feline leukemia virus infection can occur in free-living Felis silvestris. It is possible that the disease exists as a sustained infection in some wildcat populations, although the close interaction between wildcat and the domestic cat means that the latter could act as a continual source of infection.

Inhibition of gamma-glutamyl transpeptidase potentiates the nephrotoxicity of glutathione-conjugated chlorohydroquinones.

Administration of either 2,5-dichloro-3-(glutathion-S-yl)-1, 4-benzoquinone (DC-[GSyl]BQ) or 2,5,6-trichloro-3-(glutathion-S-yl)-1,4-benzoquinone (TC-[GSyl]BQ) to male Sprague-Dawley rats caused dose-dependent (50-200 mumol/kg; iv) renal proximal tubular necrosis, as evidenced by elevations in blood urea nitrogen (BUN), and in the urinary excretion of lactate dehydrogenase (LDH), gamma-glutamyl transpeptidase (gamma-GT) and glucose. Renal proximal tubular necrosis was also confirmed by histological examination of kidney slices prepared from DC-(GSyl)BQ- and TC-(GSyl)BQ-treated animals. Administration of the corresponding hydroquinone conjugates (DC-[GSyl]HQ and TC-[GSyl]HQ), prepared by reducing the quinones with a threefold molar excess of ascorbic acid, resulted in a substantial increase in nephrotoxicity. Moreover, in contrast to other glutathione (GSH)-conjugated hydroquinones, the nephrotoxicity of both DC-(GSyl)HQ and TC-(GSyl)HQ was potentiated when rats were pretreated with AT-125, an irreversible inhibitor of gamma-GT. Neither the quinone-GSH nor the hydroquinone-GSH conjugates caused any effect on liver histology or serum glutamate-pyruvate transaminase levels. The results suggest that coadministration of ascorbic acid with DC-(GSyl)BQ or TC-(GSyl)BQ decreases their interactions with extrarenal nucleophiles, including plasma proteins, and thus increases the concentration of the conjugates delivered to the kidney, and hence toxicity. Furthermore the ability of AT-125 to potentiate the nephrotoxicity of DC-(GSyl)HQ and TC-(GSyl)HQ suggests that metabolism of these conjugates by gamma-GT constitutes a detoxication reaction.