Hepatic Alkaline Phosphatase Activity Research Articles

Sub-chronic exposure to waterborne extracellular microcystin-LR impairs calcium homeostasis in rainbow trout

Fish mortality is associated with harmful algal blooms, although whether toxicity is related directly to the presence of cyanotoxins or the prevailing water chemistry remains unclear. Similarly, while planktivorous fish may be exposed to toxin through the diet, the hazard posed by waterborne extracellular toxin to carnivorous fish is less well understood. In this study rainbow trout (Oncorhynchus mykiss) were exposed for up to 28 d to waterborne microcystin-LR at nominal concentrations of 1.5 and 50 µg L−1 (measured values 2 and 49 µg L−1, respectively). The former represents the Canadian drinking water guideline, and the latter an elevated environmental level. This study hypothesised that waterborne toxin exposure would specifically impact gill function, and given the importance of this tissue in freshwater fish ion regulation, effects on plasma ions and branchial ion transporter activity would be observed. Microcystin-LR exposure resulted in a significant and persistent hypocalcaemia at the higher exposure concentration, but plasma sodium and branchial activities of the sodium/potassium ATPase, proton ATPase and calcium ATPase enzymes remained unaffected. An in vitro assessment failed to show any effect of microcystin-LR on branchial calcium ATPase activity even at exposure concentrations as high as 1000 µg L−1. A transient increase in hepatic alkaline phosphatase activity was also observed at 49 µg L−1, but there were no effects of toxin exposure on branchial or hepatic lactate dehydrogenase activity. These results suggest that microcystin-LR exposure does not have a general effect on ion regulation, but instead produces a novel and specific impact on calcium metabolism in rainbow trout, although the mechanism underlying this effect remains unknown.

Biochemical and histopathological responses in Nile tilapia exposed to a commercial insecticide mixture containing dinotefuran and lambda-cyhalothrin

The indiscriminate use of pesticides has led to an increased risk of environmental contamination and pest resistance worldwide, favoring the development of less hazardous formulations. The commercial insecticide ZEUS® (Ihara, Brazil) combining dinotefuran and lambda-cyhalothrin was recently formulated in order to meet the environmental sustainability and food security. However, little is known about the potential toxic effects of ZEUS® to aquatic species. Thus, we report, for the first time, the biochemical and histological responses in tilapia (Oreochromis niloticus) following 96 h exposure to 0.01 mg/L, 0.05 mg/L and 0.1 mg/L ZEUS®. Different biochemical endpoints, including acetylcholinesterase (AChE), gamma-glutamyltransferase (GGT) and alkaline phosphatase (ALP), were assessed as potential biomarkers of insecticide effects. Glutathione S-transferase (GST) was evaluated as a marker of phase II biotransformation, and histopathological changes were measured to indicate gill alterations following ZEUS® exposure. After 96 h exposure, ZEUS® treatment increased GST activity in the liver of fish exposed to the highest concentration, while the intermediate dose increased both renal GGT and hepatic ALP activities. These findings reflect the importance of the liver and kidneys in the detoxification of ZEUS® and highlight the need to understand further toxicity effects. Likewise, the histopathological analysis of gills provided evidence that ZEUS® caused moderate damages. Despite biomarkers alterations reported for O. niloticus following ZEUS® exposure, by comparing our findings with data on toxicity of individual compounds, the commercial ZEUS® mixture seems to present similar or even lower adverse effects on freshwater fish.

Dietary protein requirement of juvenile mud crab Scylla paramamosain

This experiment was carried out to investigate the optimal dietary protein levels for juvenile Scylla paramamosain. Six isoenergtic and isolipidic semi-purified diets were formulated with graded protein levels, and the final protein concentrations were 32.69%, 38.54%, 42.92%, 49.89%, 52.09% and 60.07%, respectively. Each diet was offered to triplicate groups of 28 mud crabs (initial weight 0.04 ± 0.001 g, carapace length 3.3 ± 0.05 mm) for 8 weeks. The results showed that the mud crabs fed diets with 32.69% and 60.07% protein had significantly lower survival than those in other treatments (p < .05). The juvenile mud crab consuming 42.92%, 49.89% and 52.09% dietary protein obtained significantly higher weight gain (WG) and specific growth rate (SGR) than other groups (p < .05). Crude protein content in whole body significantly increased with increasing dietary protein level (p < .05). Crabs fed diet with 49.89% had the lowest hepatic aspartate aminotransferase (AST) activity (p < .05). A relatively higher of hepatic alkaline phosphatase (AKP) activity was observed in groups with 42.92% and 49.89% protein diet (p < .05). The hepatic superoxide dismutase (SOD) activity significantly increased with dietary protein level increasing to 49.89% and leveled off thereafter (p < .05). The EcR mRNA expression level in the eyestalk slightly decreased with increasing protein level to 42.92%, and it then obviously up-regulated with dietary protein content further increasing to 52.09% (p > .05). According to the polynomial regression analysis, 47.06% dietary protein was required for juvenile S. paramamosain to obtain maximum growth rate.

Inclusion of microbe-derived antioxidant during pregnancy and lactation attenuates high-fat diet-induced hepatic oxidative stress, lipid disorders, and NLRP3 inflammasome in mother rats and offspring.

ObjectiveThis study aimed to evaluate the effects of microbe-derived antioxidant (MA) on high-fat diet (HFD)-induced hepatic lipid disorders in mother rats and offspring.MethodsA total of 36 female rats were randomly divided into three groups at the beginning of pregnancy: the control group (CG), HFD, and HFD with 2% MA. Mother rats were slaughtered at the first and 10th day of lactation (L1 and L10) and offspring were slaughtered at L10. The plasma and liver of mother rats, and liver of offspring were collected.ResultsThe results showed that MA reversed HFD-induced activities of inducible nitric oxide synthase (iNOS) and antioxidative enzymes in liver of mother rats and offspring. In addition, MA reduced HFD-induced lipid accumulation through decreasing the low-density lipoprotein cholesterol (LDLC) content in plasma of mother rats and improving hepatic fatty acid synthase (FAS) in mother rats and offspring. MA decreased HFD-induced hepatic alkaline phosphatase (AKP) activity in liver of mother rats and offspring. Furthermore, MA reduced HFD-activated nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome in liver of mother rats and offspring.ConclusionsMA supplementation reversed HFD-induced hepatic oxidative stress, lipid accumulation, NLRP3 inflammasome, and function in mother rats and offspring, suggesting MA can be functional ingredients to improve maternal-fetal health.

Effects of two different culture systems on the growth performance and physiological metabolism of tiger pufferfish (Takifugu rubripes)

Tiger puffer (Takifugu rubripes) is a promising species for aquaculture production because of its high commercial value and limited supply. The growth performance and physiological metabolism of subadult tiger pufferfish reared in offshore sea cage aquaculture system (OSCS) and recirculating aquaculture system (RAS) were examined in this study. Results showed that the final body weight, specific growth rate and plasm growth hormone of the female tiger puffer reared in the OSCS were significantly higher than those of the fish reared in the RAS. The condition factor and gonadosomatic index of the female tiger puffer were significantly higher than those of the male tiger puffer cultured in the OSCS and the RAS. By contrast, the hepatosomatic index, viscerosomatic index and survival rate of the tiger puffer did not significantly change. Female fish reared in the OSCS with the significantly higher hepatic alkaline phosphatase activities and lower aspartate aminotransferase compared with those of fish reared in the RAS. The hepatic pyruvate kinase and Na+-K+ ATPase activities of the female or male fish in the OSCS were significantly higher than those of the female or male fish in the RAS, respectively. The mRNA expression of somatic growth axis-related genes (igf1, igf2, igf1r, igf2r, ghr1, ghr2) of the female tiger puffer reared in the OSCS was significantly higher than that of the fish reared in the RAS. These results indicated that the growth performance and metabolic state of the female tiger puffer reared in the OSCS were better than those of the fish in the RAS, the OSCS was more suitable than the RAS for the short-term culture of subadult tiger puffer. These findings enhanced our understanding of the combined land (RAS) and sea (OSCS) alternative aquaculture model of tiger puffer.

Plasticity of energy reserves and metabolic performance of discus fish (Symphysodon aequifasciatus) exposed to low-temperature stress

A 28-day experiment was conducted to investigate the temporal variations in cellular energy allocation (CEA), oxygen consumption rate (OCR) and activities of energy metabolism related enzymes in discus fish (Symphysodon aequifasciatus) exposed to decreased water temperatures. The steady temperature decrease (1°C/day) from control (28°C) to low temperatures (24 or 20°C) significantly reduced the initial energy reserves (mainly lipids and carbohydrates) while significantly increased the energy consumption (respiratory electron transport system (ETS) activity) in the muscle tissue. Accordingly, on day 0 the CEA was slightly reduced with decreased temperatures. After 28days of cold acclimation, however, the initial differences in energy reserves, ETS activity and CEA between treatments were decreased. Concurrently, the OCR was reduced with decreased temperatures, suggesting a decreased metabolic rate. During cold acclimation, however, activities of hepatic alkaline phosphatase (ALP), lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were generally increased with decreased temperatures, indicating a compensation for the decreased metabolic rate. Linear discriminant analysis (LDA) showed that the fish exposed to 24°C only on day 14 was separated from the initial day 0 while those exposed to 20°C on both days 14 and 28 were separated from the initial day 0, with LDH and ALP activities being the two most discriminant factors. Moreover, after cold acclimation the fish exposed to 24°C showed comparable growth rates with those exposed to 28°C. These findings reveal that S. aequifasciatus could recover from the initial energy disturbances resulted from decreased temperatures. The enhanced metabolic activity in response to 24°C was well within the homeostatic range. It might be promising to culture the juvenile S. aequifasciatus at water temperature as low as 24°C.

How Ketamine Affects Livers of Pregnant Mice and Developing Mice?

It is well known that ketamine abuse can induce liver damage in adult addicts, but the effects of ketamine abuse in pregnant mothers on their offspring have received less attention. In this study, we investigated the effects of 5-day ketamine injections (30 mg/kg) to pregnant Institute for Cancer Research (ICR) mice during early gestation or mid-gestation on the aspartate aminotransferase (AST) and alkaline phosphatase (ALP) activities of the mothers and the offspring. We also looked into whether administering ketamine treatment to the mothers had any effects on the extent of fibrosis, cell proliferation and cell death in the livers of the newborns. No significant biochemical differences were found between treatment and control groups in the mothers. In the offspring, ketamine treatment mildly suppressed the gradual increase of hepatic AST activity in neonates during liver maturation. Measurements of hepatic ALP activity and lactic acid dehydrogenase (LDH) immunoreactivity revealed that ketamine treatment may lead to increased cell death. Proliferation of liver cells of the newborns was also retarded as shown by reduced proliferative cell nuclear antigen (PCNA) immunoreactivity in the ketamine groups. No obvious fibrosis was evident. Thus, we demonstrated that ketamine administration to pregnant mice suppressed hepatic development and also induced liver cell death of the offspring.

Abstract 2806: Effect of ED-71, an analogue of Vitamin D3, on intestinal neoplasia in the Apc+/Min-FCCC mouse model

Abstract Numerous studies have demonstrated an inverse relationship between circulating Vitamin D levels and risk of colorectal cancer. Although calcitriol, the most active form of Vitamin D3, exhibits promising antitumorigenic activity, its use as a chemopreventive agent has been restricted by its hypercalcemic effect at therapeutic doses. Eldecalcitol (1α,25-dihydroxy-2β-(3-hydroxypropyloxy) Vitamin D3 or ED-71), a novel analog of calcitriol, is more potent than calcitriol in stimulating bone modeling and is approved for the treatment of osteoporosis in Japan. The goal of the present study was to assess the activity of ED-71 against colorectal adenomas in a unique mouse model of spontaneous multiple intestinal neoplasia (Apc+/Min-FCCC mice). In a preliminary dose-finding study, male Apc+/Min-FCCC mice (6 weeks old, 6-7/group) were treated with vehicle (medium chain triglyceride (MCT)), calcitriol (0.25 μg/kg body weight (bw)), or ED-71 (0.2, 0.1, 0.05, 0.025, 0.0125 μg/kg bw) every other day by gavage for 6 weeks. Body weight was recorded weekly and mice were euthanized at the end of the study. ED-71 was well tolerated at all doses tested, with no significant difference in body weight observed in treated animals as compared to controls. Drug toxicity was assessed by measuring serum calcium levels by colorimetric assay (BioVision Inc.) and hepatic alkaline phosphatase (ALP) by ELISA (Antibodies-online.com). No significant effect of ED-71 on serum calcium levels and hepatic ALP activity was observed. However, because some concern exists regarding the ability of mice treated with high-dose ED-71 (0.2 μg/kg bw) to maintain their body weight long-term, ED-71 doses of 0.05 and 0.1 μg/kg bw were selected for the long-term efficacy study. Prior to entry on this study, male Apc+/Min-FCCC mice (6 weeks old) were prescreened endoscopically to determine tumor-bearing status. Mice were randomized based on tumor status (tumor-free or tumor-bearing) and body weight to one of four treatment groups: vehicle (MCT), calcitriol (0.25 μg/kg bw), ED-71 (0.05 μg/kg bw) or ED-71 (0.1 μg/kg bw). Drugs were administered by gavage every other day for 14 weeks. At the end of the study, intestinal tissue was examined grossly and tumor multiplicity was recorded. No significant effect of ED-71 on tumor multiplicity was observed in tumor-bearing mice. However, in tumor-free mice (11-13/group), treatment with ED-71 (0.1 μg/kg bw) reduced the incidence of gross tumors by 36% (P = 0.017) and colon tumor multiplicity by 30% as compared to that of controls (63.6% vs. 100%, 1.4 ± 0.4 vs. 2.0 ± 0.4, respectively). These findings suggest that ED-71 is effective in inhibiting colon tumorigenesis when given to mice prior to tumor formation. These promising data provide support for future studies to determine the potential utility of ED-71 for the prevention of colorectal cancer in high-risk subjects. (Supported by NCI HHSN261201200015I) Citation Format: Wen-Chi L. Chang, Esther Kaunga, Harry S. Cooper, Lisa Vanderveer, Jing Peng, Yongchao Zhang, Chen S. Suen, Margie L. Clapper. Effect of ED-71, an analogue of Vitamin D3, on intestinal neoplasia in the Apc+/Min-FCCC mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2806. doi:10.1158/1538-7445.AM2015-2806

Chronic Cadmium Exposure Lead to Inhibition of Serum and Hepatic Alkaline Phosphatase Activity in Wistar Rats

Alkaline phosphatase (ALP) activity in the serum and liver from rats administered with cadmium (Cd) in drinking water was studied. After metal administration, Cd showed a time-dependent accumulation in the liver, meanwhile metallothionein had a maximum increase at 1 month, remaining in this level until the end of the study. On the other hand, serum and liver ALP activity was decreased after 3 months exposure. To determine if Cd produced an inhibition on enzyme, apo-ALP prepared from both nonexposed and exposed rats was reactivated with Zn, showing 60% more activity as compared with the enzyme isolated from nonexposed rats. In vitro assays showed that Cd-ALP was partially reactivated with Zn; however, in the presence of cadmium, Zn-ALP was completely inhibited. Kinetic studies indicate a noncompetitive inhibition by Cd; these results suggest that Cd can substitute Zn, and/or Cd can interact with nucleophilic ligands essential for the enzymatic activity.

Protective Effects of Extract from Sclerotium of the King Tuber Medicinal Mushroom, Pleurotus tuberregium (Higher Basidiomycetes) on Carbon Tetrachloride-Induced Hepatotoxicity in Wistar Albino Rats.

This study was undertaken to investigate the effect of 50% ethanol extract from the sclerotia of Pleurotus tuberregium on hepatocellular integrity of albino rats treated with carbon tetrachloride (CCl4). Activities of hepatic enzymes alkaline phosphatase (ALP), alanine transaminase (ALT), and aspartate transaminase (AST) were determined in serum. Antioxidant enzyme activities, total protein, and level of malondialdehyde (MDA) were assayed in liver homogenate from treated rats. Oral administration of CCl4 (2 ml/kg body weight/day) for 7 consecutive days caused hepatotoxicity. There was a significant increase in the activity of serum ALT, AST, and ALP and the level of MDA in the liver. However, the activities of catalase and superoxide dismutase, which are antioxidant enzymes, were significantly reduced (p < 0.05). Concomitant treatment with 250 mg/kg body weight of extract and CCl4 for 7 consecutive days resulted in a significant decrease in the activity of liver marker enzymes [46% (ALT), 67.25% (AST), and 68.51% (ALP)]. These activities were further decreased [68.24% (ALT), 78.39% (AST), and 75.38% (ALP)] when the extract was increased to 500 mg/kg body weight. The activities of antioxidant enzymes, total protein, and MDA content of the liver were improved to levels that are not significantly different from the control. These findings, therefore, suggest that ethanol extract from the sclerotia of P. tuberregium has a protective action against CCl4-induced hepatotoxicity.

Determination of the lethal dose 50% (LD50) of cadmium chloride and the histopathological changes in male mice liver and kidneys

The lethal dose 50 (LD50) test involves the administration of cadmium in the form of CdCl2 to group of animals at an increasing dose in order to determine the dose that kills 50 percent of the test mice within a set of time frame (24 and 48h). The cadmium contents of liver and kidneys from dosed and control animals were determined by atomic absorption spectroscopy moreover, levels of alkaline phosphatase (ALP), in the target tissue were measured.. Male mice were dosed by 0.15ml cadmium (equivalent to the LD50 5.98 mg/kg body weight) in the form of CdCl2 via intraperitoneal injection for short-term treatment once per 24, 48 and 72h and, for long-term treatment once per week for 1, 4and 8 weeks then, sacrificed. The results showed that the effect of short-term and long-term Cd-administration on hepatic and renal Cd accumulation in the Swiss mice was found to be time dependent fashion. Atomic absorption examination showed that the Cd-content in both liver and kidneys increased significantly time dependent by 98-145% in liver and 100-300% in kidney at short-term treatment whereas for long-term treatment this increasing were 143-173% in liver and 200-370% in kidney respectively. This study also presents the histopathological effects in mice liver and kidney. Long-term Cd exposure produced damage to the entire kidney, including tubular degeneration, tubular cell apoptosis, interstitial inflammation and glomerular swelling and also resulted in liver injury including non-specific chronic inflammation and apoptosis, while it was less pronounced in short-term exposure.. Results of the biochemical profile indicated marked hepatic and renal toxic effect of cadmium reflected by the dramatic change in alkaline phosphatase (ALP) activity level, in the liver and kidney tissue. a significant increase (from100 to130%) in hepatic ALP activity, while the renal ALP activity level decreased by (from 95% to 65 %) as compared with those levels of the control in all relative periods of exposure.

Effect of dietary cadmium level on the growth, body composition and several hepatic enzymatic activities of juvenile yellow catfish,Pelteobagrus fulvidraco

The present study was conducted to investigate the effect of dietary cadmium (Cd) level on the growth, body composition and several enzymatic activities of juvenile yellow catfish, Pelteobagrus fulvidraco. The experimental diets were formulated with CdCl2·2.5H2O at levels of 0, 0.01, 0.1 and 1.0 g kg−1 diet, resulting in four dietary Cd levels of 0.25 (control), 4.92, 48.57 and 474.7 mg Cd kg−1 diet respectively. They were fed to juvenile yellow catfish (mean initial weight: 3.26±0.07 g, mean±SD) for 4 weeks. Weight gain, specific growth rate, feed intake and protein efficiency ratio tended to decline with increasing dietary Cd levels (P 0.05). Hepatic alkaline phosphatase, glutathione peroxidase and lactate dehydrogenase activities increased (P 0.05). The present study provided for the first time the toxic assessment of dietborne Cd in yellow catfish, based on growth performance and the changes in hepatic enzymatic activities for the fish species.

Combined Administration of N-Acetylcysteine and Monoisoamyl DMSA on Tissue Oxidative Stress During Arsenic Chelation Therapy

The present study deals with the therapeutic potential of combined administration of N-acetylcysteine (NAC) along with monoisoamyl DMSA (MiADMSA) against chronic arsenic poisoning in guinea pigs. Animal were exposed to 50 ppm arsenic in drinking water for 8 mo and subsequently treated for 5 consecutive days with 100 mg/kg NAC (orally) and MiADMSA (intraperitoneally), individually or in combination (50 mg/kg each). Arsenic exposure produced a significant depletion of blood delta- aminolevulinic acid dehydrate (ALAD) activity, increased the blood zinc protoporphyrin (ZPP) level, and reduced blood and liver glutathione (GSH) levels in guinea pigs. Hepatic oxidized glutathione (GSSG) and thiobarbituric acid reactive substance (TBARS) levels showed a marked increase, whereas hepatic alkaline phosphatase (ALP) activity decreased and acid phosphatase (ACP) activity increased on arsenic exposure. Significant depletion of liver transaminase activities on arsenic exposure suggests organ injury. Administration of MiADMSA, alone and in combination with NAC after arsenic exposure, was able to significantly enhance hepatic GSH and to reduce GSSG and TBARS levels compared to the arsenic control. Biochemical variables indicative of liver injury generally remained insensitive to any of these treatments. The recoveries in parameters indicative of oxidative stress were more marked in guinea pigs treated with combined administration of NAC and MiADMSA than monotherapy. Interestingly, there was a more pronounced depletion of arsenic from blood and tissues after combined treatment with NAC plus MiADMSA than MiADMSA. Blood and tissues copper, zinc, iron, and calcium concentrations showed a significant increase after arsenic exposure, which showed improvement, particularly after combined administration of MiADMSA and NAC. Based on these data, a proposal can be made that greater effectiveness in chelation treatment against chronic arsenic poisoning (i.e., turnover in the oxidative stress and removed of arsenic from the system) could be achieved by combined administration of an antioxidant (preferably having a thiol moiety) with MiADMSA.

Suppression of 7,12-dimethylbenz[alpha]anthracene-induced carcinogenesis and hypercholesterolaemia in rats by tocotrienol-rich fraction isolated from rice bran oil.

The anti-tumour and anti-cholesterol impacts of tocotrienol-rich fraction (TRF) were investigated in rats treated with the chemical carcinogen 7,12-dimethylbenz [alpha]anthracene (DMBA), which is known to induce mammary carcinogenesis and hypercholesterolaemia. DMBA administration to rats was associated with the appearance of multiple tumours on mammary glands after 6 months. Alkaline phosphatase (ALP) and glutathione-S-transferase (GST) are used as marker enzymes to monitor the severity of carcinogenesis. Although no tumours were visible on livers, hepatic ALP and GST activities of DMBA-treated rats were profoundly elevated in comparison to enzyme activities of normal control rats. Feeding of TRF (10 mg/kg body weight/day) for 6 months, isolated from rice bran oil (RBO), to DMBA-administered rats, reduced the severity and extent of neoplastic transformation in the mammary glands. Similarly, plasma and mammary ALP activities increased during carcinogenesis (95% and 43%, respectively), were significantly decreased in TRF-treated rats, whereas TRF mediated a further increase of 51% in hepatic ALP activity. TRF treatment to rats maintained low levels of GST activities in liver ( approximately 32%) and mammary glands ( approximately 21%), which is consistent with anti-carcinogenic properties of TRF. Administration of DMBA also caused a significant increase of 30% in plasma total cholesterol and 111% in LDL-cholesterol levels compared with normal control levels. Feeding of TRF to rats caused a significant decline of 30% in total cholesterol and 67% in LDL-cholesterol levels compared with the DMBA-administered rats. The experimental hypercholesterolaemia caused a significant increase in enzymatic activity (23%) and protein mass (28%) of hepatic 3-hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) reductase. Consistent with TRF-mediated reduction in plasma lipid levels, enzymatic activity and protein mass of HMG-CoA reductase was significantly reduced. These results indicate that TRF has potent anti-cancer and anti-cholesterol effects in rats.

Effect of spironolactone on cisplatin-induced nephrotoxicity in rabbits.

The effects of a single interaperitoneal dose of cisplatin (6.5 mg kg day(-1)), oral doses of spironolactone (20.0 mg kg day(-1)) for 5 days or the combined treatment (spironolactone+cisplatin) on the kidney function and liver function parameters, as well as the serum, liver and kidney cortical lipid contents were studied. The serum urea and creatinine concentrations (measured as kidney function parameters) were not altered by spironolactone treatment, but were significantly (P<0.001) elevated by cisplatin administration. However, animals exposed to both spironolactone+cisplatin revealed drastic increases in the serum creatinine and urea concentrations amounting to about four- and twofold those of cisplatin-alone treated animals, respectively. The histological examination of slides of kidneys from animals exposed to the combined drugs exhibited more extensive necrosis in the tubules compared to those from animals treated with cisplatin alone. Non of the drug treatments had any effects on the serum alanine transaminase (ALT) and aspartate transaminase (AST) levels (measured as liver function parameters) or liver protein content or hepatic alkaline phosphatase (ALP) activity. The histological examination also revealed apparently normal livers in all experimental groups. The cisplatin-induced nephrotoxicity was accompanied by hypercholesterolaemia and hyperphospholipidaemia, whereas spironolactone showed a hypocholesterolaemic effect. The concomitant treatment with both cisplatin and spironolactone significantly (P<0.05) raised the serum triacylglycerol (TAG) concentration compared to the cisplatin-alone-treated group. Both spironolactone and cisplatin administered separately or jointly caused accumulation of cholesterol and TAG in the kidney cortex with significant depletion of the liver cholesterol content. The present results indicated that spironolactone aggravates the cisplatin-induced nephrotoxicity in the rabbit.

Effect of P-Glycoprotein Modulators on Alkaline Phosphatase Activity in Cultured Rat Hepatocytes

Alkaline phosphatases (orthophosphoric-monoester phosphohydrolase, E.C. 3.1.3.1) are a group of nonspecific phosphomonoesterases located primarily in the plasma membrane of the cells in which they occur [1].It was recently demonstrated that alkaline phosphatase (ALP) concentration in different tissues is positively correlated with the extent of exchange surface per unit volume of the tissue, suggesting an association between ALP and transport systems [2]. Moreover, several groups [3,4,5] obtained evidence of an involvement of ALP in the modulation of P-glycoprotein activity in hepatocytes. The aim of the present study was to determine the putative influence of compounds known to modulate P-glycoprotein-mediated transport on hepatic ALP activity, by using primary cultured rat hepatocytes.The K_m and V_max values of ALP were determined (657.2 μM (306.8-933.1) and 32.0±1.5 nmol mg protein^–1 min^–1, respectively). Vanadate and corticosterone concentration-dependently reduced ALP activity, producing maximal reductions of 79% (100 μM) and 71% (100 μM), respectively. The IC50’s were found to be 7.9 μM (2.1-29.5 μM) and 2.4 μM (0.2-35.2 μM), respectively. Cyclosporin A, verapamil, octreotide, kaempferol, daunomycin and genistein produced a concentration-dependent increase in ALP activity. ALP activity was maximally increased to 253%, 390%, 180%, 487%, 449% and 193% of control in the presence of 100 μM cyclosporin A, 50 μM verapamil, 10 μM octreotide, 100 μM kaempferol, 100 μM daunomycin and 1 μM genistein, respectively.The results show that all P-glycoprotein modulators tested were able to significantly affect the activity of hepatic-ALP. These effects on ALP activity may contribute to the modulation of P-glycoprotein activity by these drugs.

SUBCHRONIC TOXICITY OF BENZOTHIOPHENE ON RATS FOLLOWING DIETARY EXPOSURE

The systemic and neurobehavioral effects of benzo\\[b]thiophene (routinely referred to as benzothiophene) were studied in rats following 13-wk oral exposure. Male (170 +/- 16 g) and female (146 +/- 12 g) Sprague-Dawley rats (10 animals per group) were fed diet containing 0.5, 5, 50, or 500 ppm benzothiophene for 13 wk. Control animals were given rat feed plus vehicle ( corn oil) only. No clinical signs of toxicity and neurobehavioral effects were observed using screening tests that included cage-side observations, righting reflex, open field activities, and forelimb and hindlimb grip strength. Elevated serum aspartate aminotransferase activity and bilirubin level were observed in highest dose females. Except for a statistically significant decrease in hematocrit in the highest dose males, benzothiophene exerted no marked effects on hematological parameters. Benzothiophene exposure did not result in alterations in hepatic alkaline phosphatase activity, or the typical hepatic phase I (aniline hydroxylase, ethoxyresorufin O -deethylase, pentoxyresorufin O -dealkylase, aminopyrine N -demethylase) and phase II (UDPglucuronosyltransferase, glutathione S -transferase) drug-metabolizing enzyme activities. No significant elevation in urinary ascorbic acid, protein, and N -acetylglucosaminidase activity was detected in the treated animals. Peribiliary fibrosis was the most significant histological change and occurred in the liver of females in the 50 and 500 ppm groups. Mild epithelial hyperplasia in the renal pelvis was detected in the majority of 5 and 50 ppm females, with epithelial hyperplasia in the urinary bladder observed in the 50 ppm females. In males, increased incidence and severity of mild binucleation of hepatocytes and mild thickening of the basement membrane in kidney cortex were observed at 500 ppm. Benzothiophene was not detected in the urine of high-dose animals at the termination of the experiment. Based on the kidney, hepatic, and hematocrit changes, the no-observed-adverse-effect level (NOAEL) in the diet was determined to be 0.5 ppm (0.04 mg/kg/ d) for females and 50 ppm (3.51 mg/kg/ d) for males.

CCK-8 infusion increases plasma LMW alkaline phosphatase coincident with enterohepatic circulation of bile acids.

Bile acids may facilitate the release of liver alkaline phosphatase (ALP) from its hydrophobic membrane anchor. The purpose of this study was to determine whether such a facilitatory role could be observed during the enterohepatic circulation of bile acids in dogs. Increased hepatic ALP activity was induced in four dogs by daily injections of 4 mg.kg-1.day-1 of prednisone for 10 days. Intravenous infusions of cholecystokinin octapeptide (CCK-8) were given before treatment and on treatment days 3, 5, 7, and 10 to induce gallbladder emptying and the enterohepatic circulation of bile acids. Blood samples were taken hourly for 4 h before and for 4 h after CCK-8 infusion. These showed that plasma ALP activity increased significantly only after CCK-8 infusion. Gel exclusion chromatography, polyacrylamide gel electrophoresis, and octyl Sepharose phase separation showed that the increased ALP activity was a hydrophilic, low-molecular-weight (LMW) isoform, which is consistent with phospholipase release. Histochemical staining of endogenous ALP activity showed increased ALP activity over sinusoidal surfaces of prednisone-treated dogs. There was also an increased serum-to-tissue ratio of ALP activity in the prednisone-treated dogs, suggestive of increased release of ALP into blood. It was concluded that bile acids probably play a facilitatory role in the enzymatic release of ALP from the sinusoidal surface of hepatocytes, which may be accentuated by the presence of increased amounts of ALP on the sinusoidal surface in some disease states.

Reference standards for quantification of skeletal alkaline phosphatase activity in serum by heat inactivation and lectin precipitation

Putative standards of skeletal alkaline phosphatase (ALP) (from bone, bone cells, osteosarcoma cells, and Pagetic serum) and hepatic ALP (from cholestatic serum and bile) were used to compare three methods for quantifying skeletal ALP activity in serum: heat inactivation, precipitation with wheat germ agglutinin (WGA), and precipitation with concanavalin A (Con A). All the skeletal ALP standards were similarly sensitive to heat inactivation, as were the hepatic ALP standards. Heat inactivation separated skeletal from hepatic ALP by a 50% difference in remaining ALP activities (e.g., 23% and 74% remaining skeletal and hepatic ALP activities after 30 min at 52 degrees C). Differential precipitations with WGA and with Con A were less efficient at separating skeletal from hepatic ALP (maximum differences of < 30% remaining ALP activity). Although both types of hepatic ALP standard (cholestatic serum and bile) were precipitated with similar efficiencies by WGA and Con A, the skeletal ALP standards were not (e.g., at 2.7 g/L, WGA precipitated 78-86% of the ALP activity in Pagetic serum, but only 49% of the ALP activity in extracts of human bone). These data suggest that heat inactivation is preferable to precipitation with WGA or Con A for quantifying skeletal ALP activity in serum: it better separates skeletal from hepatic ALP activity and is not sensitive to glycosyl heterogeneity.

Mobilization and Distribution of Beryllium Over the Course of Chelation Therapy with Some Polyaminocarboxylic Acids in the Rat

The efficacy of three common polyaminocarboxylic acids in the treatment of experimental beryllium intoxication was investigated in male rats. N-(2-hydroxyethyl) ethylene diamine triacetic acid (HEDTA) was more effective than calcium disodium ethylenediamine tetraacetic acid (CaNa2EDTA) in reducing the beryllium concentration of the blood, kidneys and spleen and reducing beryllium-induced inhibition of hepatic alkaline phosphatase activity. HEDTA was also most effective in reducing histopathological lesions in the liver and spleen. Compared to these two chelators, the third amino chelator, calcium trisodium diethylene triaminepenta acetic acid (CaNa3DTPA) produced severe deleterious effects in the liver and systemic toxicity. The results suggest that HEDTA is a promising chelator for beryllium toxicity while DTPA enhances the toxic manifestation of beryllium.