Experimental and human nephrotoxicity induced by ochratoxins

Ochratoxin A is a mycotoxin: a dihydroisocoumarin derivative linked to L. beta phenylalanine. Ochratoxin A is produced by a number of Aspergillus and Penicillium species. This mycotoxin is a carcinogenic, teratogenic, mutagenic and immunosuppressive substance. Ochratoxin A is a common contaminant found in a variety of foods for human nutrition as well as animal feeds. The aim of this study is to discuss nephrotoxic properties of this mycotoxin in humans. Nephrotoxicity has been reported in many animals after exposure to ochratoxin A. Porcine nephropathy due to this mycotoxin is a well known disease characterized by impairment of proximal renal function. Renal damage is also confined to the proximal tubule in other animal species. A good correlation is found between renal function abnormalities and the location of the lesions along the nephron. Of particular interest is the presence of nuclear abnormalities of the epithelial cells with pyknosis, karyorrhexis and karyomegaly. The question is to know if ochratoxin is nephrotoxic in humans. Acute nephrotoxicity seems to be very rare and we found only one case report suggesting such a possibility. We observed the occurrence of chronic renal failure in two patients with a possible responsibility of a chronic ochratoxin A intoxication. Clinical and histologic findings in these two patients were quite similar to those described in several cases of karyomegalic interstitial nephritis. Striking similarities between the changes in renal structure and function seen in ochratoxin A-induced experimental nephropathies and in Balkan endemic nephropathy suggest a common etiologic agent. This mycotoxin could be also responsible for interstitial nephropathies in North Africa.     

Synergistic antitumor activity of cisplatin and interleukin 1 in sensitive and resistant solid tumors

The antitumor activity of cis-diamminedichloroplatinum(II) (cP) and human recombinant interleukin-1 alpha (IL-1 alpha) was studied in RIF-1 and SC VII solid tumor models and in a cP-resistant subline of RIF-1 designated RIF-R1cP. In RIF-1 tumors, clonogenic cell survival after cP plus IL-1 alpha combinations was highly schedule and IL-1 alpha dose dependent. More than additive clonogenic cell kill was seen when cP was given 6 h before, but not 8 h before or at 2-6 h after IL-1 alpha. Time course studies indicated that maximal clonogenic cell killing was achieved within 4-6 h after the cP plus IL-1 alpha combination, with little or no recovery for up to 24 h. In vivo dose-response studies indicated that cP plus IL-1 alpha combinations induced more clonogenic cell kill than cP alone in all three tumor models, and analysis by the median effect principle indicated highly synergistic antitumor activity. Dexamethasone but not indomethacin inhibited the synergistic interaction. IL-1 alpha had no effect on the cytotoxicity of cP in SCC VII cells in vitro, and neither in vitro hypoxia nor in vivo ischemia, induced by clamping tumor blood supply, significantly affected cP clonogenic cell killing. Increased clonogenic cell killing was seen, however, after removal of the clamp, implicating reperfusion events, such as oxyradical stress, as a potential mechanism for increased cP cytotoxicity in SCC VII solid tumors. The data from our model systems provide a rationale for additional work to define the mechanisms of the synergistic antitumor activity of the cP plus IL-1 alpha combination and indicate that IL-1 alpha might be a useful adjunct to increase the clinical efficacy of cP-containing strategies for both sensitive and cP-resistant cancers.     

Long-term nephrotoxicity of cisplatin, ifosfamide, and methotrexate in osteosarcoma

The acute renal effects of chemotherapy are known, but long-term nephrotoxicity has rarely been investigated. The aim of the present study was to assess long-term renal function in children and adolescents who received at-risk chemotherapy, including cisplatin, ifosfamide, and methotrexate, to treat an osteosarcoma. Renal function tests [creatinine clearance, microalbuminuria, and renal excretion of sodium, potassium, chloride, calcium, magnesium (Mg), phosphorus (P), and uric acid] were prospectively performed 5.4+/-2.2 (+/-SD) years after chemotherapy (total cumulative dose: methotrexate 41+/-31 g/m2, ifosfamide 39+/-14 g/m2, cisplatin 674+/-188 mg/m2) in 18 children and adolescents. The results were compared with 13 normal volunteers matched for age and sex. Creatinine clearance, which was greater than 80 ml/min per 1.73 m2 in all patients, correlated with the total dose of ifosfamide (r=0.55, P<0.05) and cisplatin (r=0.48, P<0.05). Microalbuminuria was noted in 4 patients. Hypomagnesemia was present in 4 and hypercalciuria in 3 patients; renal excretion of P, Mg, and uric acid was higher in patients than in controls. Glomerular function was not significantly altered and only mild tubular dysfunction was present. Since renal excretion of P and Mg were increased in patients compared with normal volunteers and hypercalciuria was occasionally seen, divalent ion disorders are the most-likely potential complications.     

Interleukin 15 protects against toxicity and potentiates antitumor activity of 5-fluorouracil alone and in combination with leucovorin in rats bearing colorectal cancer

5-Fluorouracil (FUra) modulated by leucovorin (LV) is active in the treatment of colorectal cancer. Diarrhea and stomatitis are the most common dose-limiting toxicities. We have developed a model system in rats bearing a transplantable colon carcinoma sensitive to FUra therapy with dose-limiting toxicity profiles similar to what is observed in patients treated with either daily or weekly schedules of FUra plus LV. Interleukin 15 (IL-15), a cytokine that shares many biological activities with IL-2, was used at different doses (25, 100, and 400 microg/kg) and schedules (three doses before a single dose of FUra, FUra/LV daily x 5, or before each week of FUra/LV weekly x 4, or three doses before a single dose of FUra or FUra/LV daily x 5, then twice daily x 5 for a total of 11 doses) to evaluate its role in the modulation of the therapeutic selectivity of FUra alone and modulated by LV. IL-15 induced a dramatic decrease in chemotherapy-induced gastrointestinal toxicities, significant potentiation of antitumor activity, and an increased therapeutic index of FUra administered on single dose, daily x 5 and weekly x 4 schedules. In contrast, IL-2 (400 microg/kg) significantly potentiated the toxicity of FUra administered as a single i.v. push, with minimal potentiation of the antitumor activity. Taken together, the results clearly demonstrated the ability of IL-15, but not IL-2, to provide significant improvement of the therapeutic index of FUra alone and in combination with LV. The clinical relevance of the results obtained in this model system needs to be confirmed.     

Potentiation of cisplatin antitumor activity using a vitamin D analogue in a murine squamous cell carcinoma model system

In a murine squamous cell carcinoma (SCC) model, we have demonstrated that both 1,25-dihydroxycholecalciferol (1,25-D3) and the analogue 1,25-dihydroxy-16-ene-23-yne-cholecalciferol (Ro23-7553) have significant in vitro and in vivo antitumor activity. We have examined here the cell cycle effect of 1,25-D3 and Ro23-7553 on SCCVII/SF tumor cells by quantitating nuclear DNA using a detergent-trypsin method via flow cytometry analysis. Both 1,25-D3 and Ro23-7553 resulted in a significant increase of cells in G0-G1, with an accompanying decrease of cells in S phase. The ability to arrest cells in G0-G1 has been exploited by combining Ro23-7553 with the cytotoxic agent cisplatin (cis-diamminodichloroplatinum; cDDP). Using the in vitro clonogenic assay, pretreatment with Ro23-7553 for 24-48 h significantly enhanced cDDP-mediated tumor cell kill as compared to concurrent treatment with Ro23-7553 and cDDP or cDDP alone. To examine the effect of Ro23-7553 and cDDP in vivo, C3H/HeJ mice with 9-14-day SCC tumors were treated either for 3 days with varying i.p. doses of Ro23-7553 or for 7 days continuously through the use of Alzet pumps, and on the last day of Ro23-7553 treatment, cDDP (1-6 mg/kg) was administered. Using the in vivo excision tumor cell clonogenic assay, in which tumors were removed from animals 24 h after cDDP treatment and plated in a clonogenic assay, pretreatment with Ro23-7553 markedly enhanced cDDP-mediated clonogenic tumor cell kill, even at low doses of cDDP as compared to cDDP treatment alone. Similarly, a significant decrease in fractional tumor volume and increase in tumor regrowth delay was observed when animals were pretreated before cDDP with Ro23-7553 as compared to either agent alone. These results demonstrate a significant enhanced antitumor effect with Ro23-7553 pretreatment before cDDP both in vitro and in vivo and suggest that Ro23-7553 may potentiate cDDP cytotoxicity through effects on cell cycle progression.     

Pentoxifylline reduces nephrotoxicity associated with cyclosporine in the rat by its rheological properties

BACKGROUND: The goals of this study were to evaluate whether administration of pentoxifylline (POF) reduces the nephrotoxicity associated with cyclosporine (CsA) in the rat, and whether the effect of POF is related to its rheological properties. METHODS: Mean arterial pressure was measured by an intraarterial catheter. Glomerular filtration rate and renal plasma flow were determined by measuring inulin and para-aminohippurate clearances, after double-blind coadministration for 10 days of CsA (25 mg/kg/day) with either vehicle or POF (45 mg/kg every 12 hr). These results were compared with those obtained in control rats. Blood viscosity and erythrocyte deformability were also evaluated after treatment using a cone plate viscometer and a filtration method, respectively. RESULTS: No changes were observed in mean arterial pressure in both groups compared with controls. Glomerular filtration rate was significantly lower in CsA-treated rats (0.3+/-0.1 ml/min/100 g) than in control animals (0.6+/-0.1 ml/min/100 g, P<0.02). The coadministration of CsA with POF normalized the glomerular filtration rate (0.6+/-0.1 ml/min/100 g). A parallel decrease in renal plasma flow was observed in CsA-treated rats compared with controls (CsA+vehicle: 1.5+/-0.2 vs. control: 2.2+/-0.1 ml/min/100 g, P<0.02), this effect completely reversed by cotreatment with POF (3.1+/-0.2 ml/min/100 g). Blood viscosity was significantly higher in CsA-treated rats than in the control group (CsA+vehicle: 5.6+/-0.7 vs. control: 5.0+/-0.4 m x Pa x s, P<0.05). This effect was associated with a lower erythrocyte deformability (CsA+vehicle: 1.2+/-0.2 vs. control: 1.5+/-0.3 ml/min, P<0.05). These rheological abnormalities were normalized by coadministration with POF (blood viscosity: 4.9+/-0.7 m x Pa x s and erythrocyte deformability: 1.9+/-0.4 ml/min, P<0.05). CONCLUSIONS: Our results show that administration of POF prevents the nephrotoxicity associated with CsA. This beneficial effect could be related to its rheological properties.     

Huperzine A ameliorates the spatial working memory impairments induced by AF64A

Huperzine A, a novel, potent, reversible, and selective acetylcholinesterase (AChE) inhibitor has been expected to be superior to other AChE inhibitors now for the treatment of memory deficits in patients with Alzheimer's disease. We have assessed the effects of huperzine A on performance of AF64A-treated rats in the radial maze. AF64A (2 nmol per side, i.c.v.) caused significant impairment in rats' ability to perform the spatial working memory task. This behavioural impairment was associated with a significant decrease in the activity of choline acetyltransferase (ChAT) in the hippocampus. Huperzine A (0.4-0.5 mg kg-1, i.p.) significantly ameliorated the AF64A-induced memory deficit. These results suggest that AF64A is a useful agent for disrupting working memory processes by altering hippocampal cholinergic function, and such impairment can be effectively ameliorated by huperzine A.     

Effects of methylxanthine derivatives on antitumor activity and toxic side effect of adriamycin induced by inhibition of DNA biosynthesis

The antitumor activity of adriamycin (ADR) was enhanced by combination with theobromine or pentoxifylline. Theobromine increased the concentration of ADR in the tumor without any effects on that in the heart and the liver. The influence of the combination of theobromine or pentoxifylline with ADR on the lipid peroxide level (indicating the ADR-induced side effect) and on DNA biosynthesis (indicating the side effect and antitumor activity) were examined. When ADR was administered into mice, the lipid peroxide level in the liver and the heart increased. However, the combination of theobromine or pentoxifylline did not enhance the ADR-induced increment of the lipid peroxide level in the liver, and moreover, it inhibited that in the heart. The decrease of DNA biosynthesis in the liver and the heart, induced by ADR, were not enhanced by combination with theobromine or pentoxifylline. On the other hand, the combination of theobromine with ADR significantly increased the inhibition of DNA biosynthesis in the tumor. These findings indicate that the combination of theobromine or pentoxifylline with ADR have no effect on the side effects of ADR in the liver and the heart, with the increase of antitumor activity of ADR in the tumor, and it is suggested that these drugs will be of value as a biochemical modulator of ADR.     

Interleukin 6 differentially potentiates the antitumor effects of taxol and vinblastine in U266 human myeloma cells

OBJECTIVE: To assess past care practices of neurologists and obstetricians to identify areas in which practice patterns differ from currently accepted optimal care. METHODS: Retrospective chart review of 155 women identified as having a diagnosis of epilepsy (or seizure disorder) who had been pregnant any time between January 1988 and December 1995 and were admitted to Stanford University Hospital for delivery. A total of 161 pregnancies (132 women) were selected for study. RESULTS: An obstetrician was seen at some point during the pregnancy in 99% of the pregnancies, whereas a neurologist was seen at least once in only 64% of the pregnancies. In the 3 months before conception, an obstetrician was seen in 5% of the pregnancies and a neurologist was seen in 15%. Seventy-five percent of the patients taking antiepileptic medication and 65% of the untreated patients had documentation of folate supplementation at any time during pregnancy. Vitamin K supplementation in the final month of pregnancy was documented for only 41% of those receiving antiepileptic drugs. In over one-third of the pregnancies the mother did not have a maternal serum alpha-fetoprotein measure documented and a similar percentage did not receive genetic counseling. Monitoring of the maternal serum concentration of the non-protein-bound fraction of the prescribed antiepileptic drugs was not documented. CONCLUSIONS: We identified specific omissions of appropriate vitamin supplementation, genetic counseling, and drug level monitoring. Educational efforts should be targeted to improve the management of pregnancy in women with epilepsy.     

The antioxidant LY 231617 ameliorates functional and morphological sequelae induced by global ischemia in rats

In this study the effect of LY 231617, an antioxidant, on spatial learning deficit and on neuronal damage following transient cerebral ischemia was evaluated. Global ischemia was induced by four-vessel-occlusion (4VO) for 20 min in rats. LY 231617 (20 mg/kg i.p.) was administered after onset of reperfusion. One week after surgery spatial learning was tested in the Morris water maze. LY 231617 reduced the increase in escape latency and in swim distance induced by 4VO. Neuronal damage in the CAI sector of the hippocampus produced by 4VO was significantly attenuated by LY 231617. The present data demonstrate that posttreatment with LY 231617 exerts a protective effect on hippocampal neuronal damage and deficits in spatial learning induced by 4VO.     

Hypolipidemic activity of boronated nucleosides and nucleotides in rodents

Base-boronated nucleoside and phosphate-boronated nucleotides were potent hypolipidemic agents in rodents, lowering both serum cholesterol and triglyceride levels. Rat VLDL and LDL cholesterol levels were generally reduced and HDL cholesterol levels were significantly elevated after 14 days dosing at 8 mg/kg/day. Tissue cholesterol, triglyceride and phospholipid levels were reduced by selected derivatives. Increased fecal excretion of lipids did not appear to be a mechanism by which these derivatives lowered serum lipids in rodents. Rather, the agents suppressed appetite and reduced the activities of rate-limiting enzymes for de novo lipid synthesis, specifically cytoplasmic acetyl CoA synthetase, squalene synthetase, and phosphatidylate phosphohydrolase with IC50 values of approximately 10(-5) m.     

Chronic treatment with losartan ameliorates vascular dysfunction induced by aging in spontaneously hypertensive rats

OBJECTIVE: To evaluate the effects of prolonged treatment with losartan on endothelium-dependent and endothelium-independent relaxations of aortic rings from adult and senescent spontaneously hypertensive rats, and to clarify whether these effects were due to specific mechanisms of the drug or a consequence of its blood-pressure-lowering action. MATERIALS AND METHODS: Adult (aged 5 months) and senescent (aged 20 months) male spontaneously hypertensive rats were treated for 12 consecutive weeks with 10 mg/kg per day losartan. Systolic blood pressure and plasma concentration of nitrates were evaluated. We studied endothelium-dependent and endothelium-independent relaxations and response to angiotensin II of aortic rings from rats of each group. The direct effects of angiotensin II type 1 receptor antagonism on vascular reactivity of aortic rings from untreated adult and senescent rats that had been incubated beforehand with losartan were also studied. RESULTS: Losartan treatment comparably reduced blood pressure and increased plasma concentration of nitrates in rats of both age groups. Responses to acetylcholine and sodium nitroprusside were lower for rings from senescent than they were for rings from adult rats. Constrictor responses to angiotensin II were higher for rings from senescent than they were for rings from adult rats. Treatment with losartan increased the magnitude of relaxations in response to acetylcholine for rings from rats in both groups, but increased the magnitude of relaxations in response to nitroprusside only for rings from senescent spontaneously hypertensive rats. Incubation beforehand of aortic rings from untreated rats with losartan enhanced magnitude of relaxations in response both to acetylcholine and to nitroprusside only for rings from senescent spontaneously hypertensive rats. CONCLUSIONS: The consequences of aging for endothelium-dependent and endothelium-independent relaxations of rings from spontaneously hypertensive rats are ameliorated by losartan treatment, suggesting that angiotensin II plays a role via type 1 receptors. The effects of losartan on senescent spontaneously hypertensive rats were due not only to its blood-pressure-lowering action but also to the blockade of specific mechanisms derived from angiotensin II type 1 receptor antagonism, which might involve an increase in availability of NO.     

Histology and enzymatic activity in the postnatal development of limb muscles in rodents

The present work examines how increases in spontaneous motor capabilities during postnatal development are reflected in enzymatic activity and the histology of hindlimb muscles of the dormouse (Eliomys melanurus), the jird (Meriones tristrami), the vole (Microtus socialis), and the spiny mouse (Acomys cahirinus). The precocial neonate of the spiny mouse had the most advanced developmental state of young myofibers with striations as early as 1 week after delivery. At the same age, the altricial neonate vole had less developed muscles compared to the spiny mouse, but was more mature compared to other altricial species. The dormouse was the least developed, with numerous myoblasts and few myotubes at 1 week after delivery. These differences in myogenic development were conspicuous throughout postnatal development. Similar differences between the species were also evident at the biochemical level, as measured in the kinetics of activity of the enzyme creatine-phosphokinase immediately after delivery. On postnatal day 7, the creatine-phosphokinase level in the spiny mouse was fourfold higher than in the dormouse or vole. The enzymatic activity of acid phosphatase decreased during the first week postdelivery in the spiny mouse while peaking in the first, second, and third week in the jird, vole, and dormouse, respectively. These results support the notion that precocial species undergo certain developmental stages in utero, whereas, the same stages commence in altricials only postnatally. For the tested altricial species, the results illustrate that limb muscles in the vole, which displays more basic gaits, mature before limb muscles of the jird and dormouse, which display more specialized gaits.     

Association of macrophage activation with antitumor activity by synthetic and biological agents

Treatment of normal BALB/c mice i.p. with a number of adjuvants, including pyran copolymer, the copolymer of polyinosinic and polycytidylic acids, Bacillus Calmette-Guérin, glucan, and dextran sulfate, rendered macrophages nonspecifically cytostatic for syngeneic tumor cells. Macrophage activation was highly dose dependent. The validity of the inhibition of DNA synthesis assay for measuring macrophage-induced cytostasis of target cells was proven by demonstrating a concurrent decrease in RNA synthesis and a reduction in viable tumor cell number. Moreover, conditioned supernatants from pyran-activated macrophages did not significantly decrease [3H]thymidine incorporation by freshly added leukemia cells. Biological or synthetic agents that activated macrophages were generally effective systemic antitumor agents against the M109 lung carcinoma. Drugs that did not activate macrophages, such as typhoid vaccine, tilorone, levamisole, WY-13876, and thymosin, were ineffective in prolonging the life of tumor-bearing mice. Pyran treatment i.p. was the most effective antitumor adjuvant in two separate tumor models, and suppression of tumor growth appeared to be related not only to an increase in macrophage tumoricidal function, but also to a larger influx of macrophages responding at the tumor site.     

Synthesis, cytotoxicity and antitumor activity of some new simplified pyrazole analogs of the antitumor agent CC-1065. Effect of an hydrophobic group on antitumor activity

Three simplified pyrazole analogs (7-9) of the antitumor agents CC-1065, were synthesized. In in vitro assays, against L1210 cell lines all derivatives showed a cytotoxicity in a pM range, values close to the natural target compound (+)-CC-1065. In in vivo tests, against disseminate L1210 leukemia cells, synthesized compounds showed a good potency (O.D. 300 micrograms/Kg) but no activity. These observations further validate the effect of the hydrophilic and/or hydrophobic characteristics of the substituents present on the molecules, confirming the relevance of this phenomena on in vivo activity. In fact in this case the increase of hydrophobic characteristics of the molecules produce the loss of activity, probably due to a worse bioavailability of the drugs in animals.     

Ethanol exposure potentiates fosB and junB expression induced by muscarinic receptor stimulation in neuroblastoma SH-SY5Y cells

Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) have been shown to play a role in host defense and pathogenesis of chronic HCV infection. Our aim was to test the hypothesis that intrahepatic HCV-specific CTL activity may impact subsequent response to interferon alfa (IFN-alpha) therapy. Of the 37 patients that we have prospectively evaluated for HCV-specific CTL activity in liver, 21 received IFN therapy, and 19 completed a 6-month course and attended 6 to 18 months of follow-up. Intrahepatic CD8+ cells were isolated from liver biopsy tissue and tested against target cells expressing HCV antigens to determine intrahepatic CTL activity. The relationship between treatment response and HCV-specific CTL activity and other factors known to associate with response (genotype, viremia, histology) was analyzed. HCV-specific CTL activity was detected in 9 of 21 patients (and 9 of 19 who completed therapy). After 6 months of IFN therapy, 8 of 19 (42%) patients had normal serum alanine transaminase (ALT) (complete responders). After 18 months of follow-up, only 3 patients (16%) had a sustained biochemical response. Of the 9 patients with detectable HCV-specific CTL activity in their liver before treatment, 7 (78%) developed a complete response. In contrast, only 1 of the 10 patients with no detectable HCV-specific CTL activity developed a complete response to IFN (P < .01). In 6 of 8 patients with a complete response, including the 3 sustained responders, the CTL response appeared to be directed predominately to the HCV core region. These data suggest that the host immune response, particularly that mediated by CD8+ CTL, may be important in determining the outcome of IFN therapy for chronic HCV infection. Further understanding of the mechanism of action of IFN should impact the design of better therapeutic strategies against chronic HCV infection.     

Tumor necrosis factor alpha potentiates the increase in cytosolic free calcium induced by bradykinin in guinea-pig tracheal smooth muscle cells

The ability to respond to bradykinin (BK) by a bronchospasm is one of the most intriguing characteristics observed in asthmatic patients but not in healthy subjects. The molecular basis of this sensitivity is not yet known. Therefore, we studied the effect of BK, and its putative modulation by tumor necrosis factor alpha (TNF alpha), on cytosolic free Ca2+ concentration ([Ca2+]i) in fura-2-loaded tracheal smooth muscle cells in culture. BK induced a concentration-dependent rise in [Ca2+]i at concentrations between 10(-13) and 10(-11) M which was mediated via BK receptors of the B2 subtype. The net increase in [Ca2+]i induced by 10(-12)M BK was 478 +/- 52 nM. Pre-treatment of the cells with TNF alpha (10 ng/ml) for 24 h significantly potentiated this net increase in [Ca2+]i to 956 +/- 154 nM. The presence of anti-TNF alpha antibodies inhibited this potentiation. These results show that TNF alpha is able to interact with airway smooth muscle cells which suggests the existence of receptors for TNF alpha on these cells. The study, in vivo, of such interaction should help to further elucidate the mechanisms involved in airway hyperresponsiveness.