Structure-activity relationship for two lipoxygenase inhibitors and their potential for inducing nephrotic syndrome.

In a study of structure-activity relationship with drug-induced nephropathy two lipoxygenase inhibitors, the N-hydroxyurea derivative 70C ((E)-N-{3-[3-(4-fluorophenoxy) phenyl]-1-(R, S)-methylprop-2-enyl}-N-hydroxyurea) and the N-hydroxamic acid analogue 360C ((E)-N-{3-[3-(4-fluorophenoxy) phenyl]-1-(R, S)-methylprop-2-enyl}-N-hydroxamic acid), were administered to rats. 70C and 360C were dosed to female Wistar rats at 100 mg/kg po daily for 7 days. Another group of rats was given a single intravenous bolus dose of puromycin aminonucleoside (PAN) at 100 mg/kg. Urine samples were collected from all groups during the study and plasma samples were collected after 7 days. Kidneys were excised and fixed for examination by electron microscopy. 70C- and PAN-treated groups both showed early changes in the glomeruli, in which the visceral cells appeared enlarged and showed varying degrees of foot process loss. This foot process loss was associated with decreases in total plasma protein and albumin and increases in the plasma cholesterol, triglycerides, creatinine, and urea were recorded. Marked proteinuria was observed in both the 70C and PAN groups. The foot process loss together with increased proteinuria, hypoalbuminemia, hypercholesterolemia, and lipemia are all characteristic of the human condition, Minimal Change Nephrotic Syndrome. All the biochemical and morphological investigations showed that 360C-treated rats were similar to the control group, suggesting that the hydroxyurea moiety of 70C is responsible, either directly or indirectly, for the induction of the nephrotic syndrome seen in rats.

Nephrotic syndrome associated with N-hydroxyureas, inhibitors of 5-lipoxygenase.

The N-hydroxyurea derivatives 70C ((E)-N-[3-[3- (4-fluorophenoxy)phenyl[-1-(R,S)-methylprop-2-enyl]-N-hydroxyurea) and its (R) 225C and (S) 404C enantiomers, which were being developed as 5-lipoxygenase inhibitors for the treatment of certain allergic and inflammatory conditions, were found to cause severe glomerulonephropathy in the rat. The lesion appeared to be of greater severity in female rats compared with male rats. In addition, 70C and 225C treated animals appeared more severely affected than 404C treated animals. Detailed examination of the lesion in animals dosed with 225C showed that there was a clear relationship between the onset of the lesion and the dose given, i.e. the higher the dose the sooner the lesion developed. The earliest changes detected in the kidney by transmission electron microscopy were noted in the glomeruli, in which the visceral cells appeared enlarged and showed varying degrees of foot process loss. In the more advanced lesion, the degree of foot process loss became more obvious and changes in the kidney tubules were seen by light microscopy. The morphological changes were mirrored by a dose-related increase in water consumption, an increased kidney to body weight ratio and gastrointestinal oedema, suggesting impaired renal function. Shortly after the onset of foot process loss, decreases in the total plasma protein and albumin and increases in the plasma cholesterol, triglycerides, urea and creatinine were recorded. These changes, particularly the foot-process loss, together with increased proteinuria, hypoalbuminaemia, hypercholesterolaemia and lipaemia, are all characteristic of "minimal change nephrotic syndrome". Because of the serious nature of the kidney lesion caused by these N-hydroxyureas in the rat, it was considered that it precluded their development as therapeutic agents for use in man.

Paradoxical fate and biological action of peroxynitrite on human platelets.

Peroxynitrite (ONOO-), which is formed from the reaction of nitric oxide (NO) and superoxide (O2-), has been suggested to be responsible for some of the cytotoxic effects of these molecules. When protonated, ONOO- gives rise to hydroxyl (OH.) and nitrogen dioxide (NO2) radicals, which are capable of inducing tissue damage. We have investigated the effects of ONOO- on human platelets in vitro in order to explore the potential of this oxidant to contribute to tissue damage. ONOO- caused aggregation of washed platelets and reversed the inhibition of aggregation induced by S-nitroso-N-acetyl-DL-penicillamine (SNAP), prostacyclin, and indomethacin. However, in platelet-rich plasma, ONOO- not only did not possess proaggregatory properties but acted as an inhibitor of platelet aggregation. This reversal of the aggregatory effect of ONOO- could also be achieved in washed platelets by adding low concentrations of plasma, human serum albumin, or glutathione and was inhibited by hemoglobin. An analysis of the reaction products of ONOO- and glutathione revealed the presence of both NO and S-nitrosoglutathione in quantities sufficient to account for the antiaggregatory effects observed. Thus the fate and therefore the actions of ONOO- in biological systems are critically dependent on the biological environment in which this oxidant is present.

The role of lysosomes in hyaline droplet nephropathy induced by a variety of pharmacological agents in the male rat.

The male rat is prone to hyaline droplet formation in renal proximal tubular cells. Several unrelated pharmaceutical agents exacerbate the formation and accumulation of these droplets. Where the loading of the proximal tubular cells is marked it gives rise to increased cell turnover and a hyaline droplet nephropathy develops. Cytochemical procedures, have confirmed that this accumulation of hyaline droplets represents an increase in the size and number of secondary lysosomes involved in protein uptake and metabolism. This predisposition of the male rat to develop hyaline droplet nephropathy relates to (1) the large amounts of the low-molecular-weight protein alpha 2U globulin in the glomerular filtrate, (2) the resistance of the globulin to proteolysis, and (3) the low protease activity in the proximal tubule lysosomes. The current data would suggest that the pharmacological agents, which cause the nephropathy, exert their effect by reducing the proteolytic breakdown of alpha 2U globulin in the proximal tubule lysosomes. This results in the overloading of a system which is already operating near maximal load. Female rats, and all other species excrete only small amounts of alpha 2U globulin or similar proteins, which are more easily hydrolyzed. Thus it is argued that the type of hyaline droplet nephropathy induced by these pharmacological agents is unique to the male rat and of little relevance to man.

An evaluation of the putative human mammary tumour retrovirus associated with peripheral blood monocytes.

The primary aims of this study were purification and molecular cloning of a putative retrovirus designated human mammary tumour virus (HMTV). However, our preliminary unpublished data of negative reverse transcriptase (RT) activity in ostensibly 'infected' cells led us to re-examine the evidence for this virus; namely multinucleate giant cell (MNGC) formation and RT activity in cultured blood monocytes from breast cancer patients versus benign breast tumour and normal control subjects. MNGCs from by fusion of monocytes and we estimated the total number of cell fusions which had occurred after 10 days of culture in vitro by counting cells with two, three, four and five or more nuclei (n) and by measuring the density of adherent mononuclear cells for each subject studied. We found no clear-cut difference in MNGC formation between the three subject groups. Moreover, a substantial number of cultures, encompassing the three groups, showed far more MNGCs per 10(5) monocytes than previously reported. Various parametric and nonparametric statistical analyses were performed on the multinucleate cell data and only one parametric test, which utilised the density of monolayers as a co-variate, showed a statistically significant difference at the 5% level between the breast cancer and the normal subject groups. We observed marked subject-to-subject variation in multinucleate cell formation and we suggest that the evidence for a difference between the breast cancer and the normal groups is marginal. Further, MNGC formation by breast cancer monocytes may not be attributed to the presence of a retrovirus since 5'-Azacytidine (AZA), an agent known to stimulate replication of latent retroviruses showed no effect on the MNGC formation. In addition, culture supernatants from the three groups were assayed for RT activity and no test sample gave a significant signal above background. Preliminary transmission electron microscopy analysis failed to identify viral particles in MNGCs.

Induction and exacerbation of hyaline droplet formation in the proximal tubular cells of the kidneys from male rats receiving a variety of pharmacological agents.

Kidneys from male and female Wistar rats dosed with 1 of 3 chemically unrelated pharmacological agents, a pyrazoline BW540C, a naphthoquinone BW58C and the levoisomer of tetramisole, levamisole or the light hydrocarbon Decalin, were examined by light and electron microscopy. Paraffin histology showed that all 4 agents induced and exacerbated hyaline droplet accumulation in the renal proximal tubular cells of the male rats. Resin histology at both the light and electron microscope level, along with cytochemical procedures for acid phosphatase and the protein 'alpha 2U globulin', helped further in the characterisation of these cytoplasmic inclusions. These techniques confirmed that the accumulation of hyaline droplets seen by paraffin histology represented an increase in the size and number of secondary lysosomes which have been shown to be involved in protein uptake and metabolism. Time course studies showed that increased numbers of small dense lysosomes appear first, which then increase in size, presumably by fusion. Crystalloid bodies form in these large lysosomes eventually giving rise to rectilinear bodies. The accumulation of these protein laden secondary lysosomes took place primarily in the cells of the S1 and S2 segments of the proximal tubules. In extreme cases of lysosomal accumulation however, loading of the S3 segments was noted. In tubules where cellular inclusion loading was heavy, there was evidence of increased cell turnover. The kidneys of female rats dosed with any one of the 4 agents appeared normal.

Isolation and washing of human platelets with nitric oxide.

Nitric oxide (NO) was compared with prostacyclin as an inhibitor of the activation of human platelets during isolation, washing and storage. The use of NO throughout the procedure prevented the activation of platelets. The morphology and behaviour of NO-washed platelets was similar to that of prostacyclin-washed platelets when stored at 4 degrees C for up to 24 h. Prolonged storage resulted in deterioration of the platelets which occurred earlier in the NO-washed than in the prostacyclin-washed platelets. The protective effect of NO was potentiated by the selective cGMP phosphodiesterase inhibitor M & B 22948, suggesting that it is mediated by the activation of guanylate cyclase.

The effects of levamisole on urinary enzyme measurements and proximal tubule cell inclusions in male rats.

A markedly increased incidence of large angular secondary lysosomes was observed in the proximal tubular cells of male Wistar rats dosed orally with levamisole, 75 mg/kg body weight for 15 days. These inclusions were similar in appearance to those previously observed in male rats treated with decahydronaphthalene. Urinary enzymes were measured throughout the study, and of these enzymes lactate dehydrogenase and N-acetyl-beta-D-glucosaminidase activities were higher on days 9 and 13 for rats dosed with levamisole in comparison with control animals. Urine volumes increased for the levamisole treatment group, but no treatment related changes of urine protein output were found.

Adhesion of human platelets to collagen in the presence of prostacyclin, indomethacin and compound BW 755C.

Prostacyclin (1 ng to 2 micrograms per ml), which effectively inhibits platelet secretion and aggregation, does not affect adhesion of a proportion of platelets (10-38%) to collagen (50-100 micrograms/ml). Adhesion is not detectable by changes of light transmission (as measured in the optical aggregometer) and is not affected by inhibitors of cyclooxygenase and lipoxygenase enzymes such as indomethacin and compound BW 755C. This adhesion is independent of the collagen concentration (50-400 micrograms/ml) and the incubation time (5-20 min). This suggests that adhesion to collagen is related to a specific platelet population. Adhesion in the presence of prostacyclin, indomethacin and BW 755C occurs in parallel with the formation of a limited amount of phosphatidic acid. Under those conditions it is also possible to observe some phosphorylation of a 40,000 dalton protein which is a substrate for protein kinase C activity. Phosphorylation of the 20,000 dalton protein, or myosin light chain, is less evident. Chlorpromazine (25-100 micrograms/ml) inhibited the adhesion of platelets to collagen, but propanolol (0.5-4 microM) was inactive. The adhesion of platelets to collagen in these experiments parallels the formation of a fraction of phosphatidic acid and 40,000 dalton protein phosphorylation, which are independent of the increased levels of platelet cyclic-AMP induced by high concentrations of prostacyclin. It is also independent of the formation of cyclooxygenase or lipoxygenase products.

Myeloperoxidase activity as a quantitative marker of polymorphonuclear leukocyte accumulation into an experimental myocardial infarct--the effect of ibuprofen on infarct size and polymorphonuclear leukocyte accumulation.

The accumulation of polymorphonuclear leukocytes (PMN) into a canine occlusion/reperfusion model of myocardial infarction (MI) has been quantitated using a spectrophotometric assay for the PMN-specific enzyme myeloperoxidase (MPO). In anaesthetized beagle dogs subjected to 1 h of occlusion of the left anterior descending coronary artery followed by coronary reperfusion for 1 h, MPO activity was found in fourfold greater amounts in infarcted myocardium as opposed to normal myocardium. MPO activity in infarcted myocardium increased with coronary reperfusion in a time-related manner, the greatest accumulation occurring at 5 h of coronary reperfusion. The time-related increase in MPO activity in ischemic myocardium was closely correlated with the observed accumulation of PMN as assessed by histological sectioning, thus supporting the specificity of the method. Pretreatment of dogs with ibuprofen (12.5 mg/kg i.v.) did not alter the MPO content and therefore PMN invasion of infarcted myocardium, a finding confirmed by light microscopy. However, pretreatment with ibuprofen increased myocardial infarct size (from 11.5 to 37.4%, p less than 0.01) and increased the incidence of haemorrhagic infarction and ventricular fibrillation. In conclusion, our studies demonstrate that MPO can be used as a sensitive and quantitative marker of PMN accumulation into an evolving MI. Furthermore, we have demonstrated that PMNs rapidly infiltrated injured myocardium in a time dependent manner and this accumulation was unaffected by ibuprofen pretreatment.

An ultrastructural study of stored human platelets after washing using prostacyclin.

The morphology of washed human platelets stored at 4 degrees C has been examined over a period of 96 h. Platelets prepared using prostacyclin showed good preservation of their internal structure. These platelets showed no signs of activation when stirred in an aggregometer and only small reversible aggregates were formed during storage. In contrast, platelets prepared using a more conventional method, which does not use prostacyclin, showed poor preservation of internal structure. Storage and stirring of these platelets resulted in activation and morphological deterioration. These observations support our previous finding that prostacyclin prolongs the viability of washed human platelets in vitro.

The accumulation of an aryloxylkylamidine (501C) and 5-hydroxytryptamine in human polymorphonuclear leucocytes: a quantitative electron microscopy study.

The uptake of anilino-N-2-m-chlorophenoxypropylacetamidine (501C) and 5-hydroxytryptamine (5-HT), in human polymorphonuclear leucocytes (PMN) was investigated by quantitative ultrastructural autoradiography. The major concentration of both drugs was in the cytoplasmic granules, 71.5 +/- 6.8% for [3H] 501C, and 68.75 +/- 6.1% for [3H]5-HT. Lesser quantities of both drugs were associated with the nucleus; 18.1 +/- 4.8% for [3H] 501C and 23.0 +/- 4.2% for [3H] 5-HT. Only small amounts of activity were recorded at other sites. The data suggests that there may be common binding sites for 501C and 5-HT in PMN. Furthermore the concentration of 501C in PMN granules may also account for the damaging effect of high concentrations of the drug on PMN.

The uptake of mepacrine by horse polymorphonuclear leucocytes in vitro.

The uptake of mepacrine by isolated horse polymorphonuclear leucocytes (PMN) was measured using spectrophotofluorimetry. Two phases of uptake were observed, the first, rapid fraction, essentially complete by 10 min, and a second, slow fraction, which was still proceeding after 60 min. The appearance of mepacrine within the PMN was also visualized by fluorescence microscopy. Discrete yellow points of fluorescence were observed in the cytoplasm of PMN within 30 s. These discrete points corresponded both in size and number to the PMN granules. After 5 min, the nuclei showed faint fluorescence which increased in intensity with time. It was concluded that the accumulation of mepacrine by horse PMN consists of at least two phases, an initial rapid component which represents uptake of the drug through the plasma membrane into the cytoplasmic granules, and a subsequent slower component, possibly representing nuclear binding.

Kanamycin induced ototoxicity in the laboratory rat. A comparative morphological and audiometric study.

Two comparative experiments on the behavioural, audiometric and histological effects of kanamycin-induced cochleotoxicity in the Wistar rat are reported. In the first experiment kanamycin 400-1,500 mg/kg/day was injected subcutaneously for 20 days and the morphological damage to the organ of Corti assessed. In the second experiment the progression of damage to the organ of Corti was examined in animals given kanamycin 700 mg/kg/day for 4-20 days. Behavioural audiometric studies of threshold shift were undertaken throughout both experiments. In the first study, all the animals were killed after a recovery period of 20 days from the last injection, i.e., day 40, and in the second study groups of animals were killed at 4-day intervals between days 4 and 20 of dosing. One cochlea from each animal was critical point-dried, dissected to expose the organ of Corti and examined by scanning electron microscopy. A vertical section through the contralateral cochlea was examined by light microscopy. The results of the morphological examination of the cochleas were collated with the behavioural audiometry. The morphological damage to the organ of Corti followed a stereotyped pattern of degeneration, the extent of which appeared to be determined both by the number and concentration of the kanamycin administrations. The collateral audiometric examinations indicated that extensive damage had taken place before a shift in the behavioural auditory threshold could be detected by observation of the Preyer reflex.

Improved morphological techniques for screening potentially ototoxic compounds in laboratory animals.

A technique is described for morphological examination of the organ of Corti which can be used in routine toxicological studies. This involves the dissection of a critical point-dried cochlea and examination of a surface preparation of the entire organ by scanning electron microscopy. The procedure is simple and far less time consuming than that previously used for light microscopy. Quantitative estimates of damage induced in the organ of Corti either by the administration of ototoxic compounds or as a response to noise-induced or age-related changes can be obtained. Details are given for the application of this technique to two species commonly used in toxicity studies. With minor modifications it could be applied to many other species of animal.

Prostacyclin: a solution to some problems of extracorporeal circulation. Experiments in greyhounds.

In cardiopulmonary-bypass experiments in greyhounds the effects of adding prostacyclin, prostacyclin plus heparin, and heparin alone to the extracorporeal circulation were compared. With heparin alone platelet count and function were reduced, the pressure differential across the arterial filters rose, platelet deposits were found on the arterial filters, and plasma-fibrinogen levels fell. Plasma from these dogs was toxic to fetal-mouse hearts in culture. With prostacyclin alone, fibrinogen levels fell, but the platelets were preserved. With a combination of prostacyclin and heparin, platelet count and function were maintained, there was no consumption of fibrinogen, and there was little deposition on the arterial filters.