Supernatant Of Liver Homogenate Research Articles

Electrochemical Activation of Metabolic Enzymes in Human Liver S9 Fractions

Human liver subcellular fractions such as liver microsomes, liver cytosol fractions, or S9 fractions are widely used in in-vitro assays to predict liver metabolism. The S9 fractions are the supernatants of human liver homogenates consist of both human liver microsomes and the cytosol. Therefore, these fractions contain majority of cytochrome P450 (CYP) enzymes and soluble phase II enzymes like glucuronosyltransferases, N-acetyl transferases, sulfotransferases, and glutathione-S-transferases. Here we report the direct electrochemistry and biocatalytic features of redox active enzymes impregnated in S9 fractions. We investigated the electrochemical properties of S9 films by immobilizing them onto a high purity graphite electrode and performing cyclic voltammetry in anaerobic (Ar saturated) and aerobic (O2 saturated) conditions. The electron transfer between the S9 film and the high purity graphite electrode was observed at the formal potential of -0.48 V, with respect to Ag/AgCl reference electrode, which confirmed the activation of CYP450 reductase enzyme in the film. These films also demonstrated a catalytic oxygen reduction with a maximum current density of ~0.5 mA cm-2 at the scan rate of 0.050V/s. We investigated the CYP activity in the S9 biofilm for phase 1 metabolism using diclofenac hydroxylation as a probe reaction and could able to identify metabolic products using liquid chromatography-mass spectrometry (LC-MS) technique. Eventually, we envision to develop a platform to perform both phase I and II metabolic reactions in a single electrochemical assay.

Sevoflurane attenuates hepatic ischemia reperfusion injury by the miR-122/Nrf2 pathway

BackgroundSevoflurane can protect organs from ischemia-reperfusion (IR) injury, but the mechanism is still unclear. MicroRNA-122 (miR-122) is a liver-specific microRNA (miRNA) and regulates liver function. Therefore, this study aims to elucidate the relationship between the protective effect of sevoflurane and miR-122 in liver IR injury.MethodsWistar rats were divided into the following groups: sham, IR, IR + sevoflurane, IR + miR-122 antagomir, and IR + miR-122 antagomir + sevoflurane. Hematoxylin and eosin (H&E) staining and Suzuki score were used to evaluate the pathological damage of the liver. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-10 in the serum and the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and nitric oxide (NO) in the liver homogenate supernatant were detected by using the corresponding kit. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) and flow cytometry was applied to evaluate the apoptosis of liver tissues. The expression of nuclear factor E2-related factor 2 (Nrf2), miR-122, p53, and HO-1 in liver tissue was evaluated by using immunohistochemistry, qRT-PCR, and western blot as needed.ResultsCompared to the IR group, the sevoflurane post-treatment or miR-122 antagomir groups showed improved liver injury, decreased Suzuki score, inhibited the levels of AST, ALT, LDH, MDA, NO, TNF-α, IL-1β, and IL-6, increased levels of SOD, IL-10, and inhibited hepatocyte apoptosis. Regarding the molecular mechanism, sevoflurane post-treatment fostered the expression of HO-1, promoted the transport of Nrf2 from cytoplasm to the nucleus, and decreased the expression of miR-122 and p53. The combined use of miR-122 antagomir and sevoflurane enhanced the protective effect of miR-122 antagomir in liver injury in IR rats.ConclusionsSevoflurane protected the liver from IR damage by regulating the miR-122/Nrf2/HO-1 pathway.

Methanol Stem Bark Extract of Ficus platyphylla Protects Against Carbon Tetrachloride-Induced Liver Damage in Wistar Rats

Ficus platyphylla (Gutta percha tree) is used in Nigerian traditional medicine for treating pain and psychosis. This study evaluated the protective effect of methanol stem bark extract of Ficus platyhylla (MEFP) against carbon tetrachloride (CCl4)-induced liver injury in Wistar rats. Rats were allotted into six groups namely normal control, negative control, standard drug (silymarin) and extract treated (100, 200 and 400 mg/kg p.o. respectively) groups. Treatment was administered for 7 days. Animals in groups II-VI received a single dose of CCl4 (1:1) p.o. in olive oil at 1.5 ml/kg body weight after last treatment. Experimental rats were sacrificed after 24 hours of CCl4 administration and blood samples collected for determination of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities. Supernatant of liver homogenate was used to determine activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx). Results indicate increased activities (p < 0.05) in CAT, SOD and GPx in samples of MEFP treated rats while levels of ALT and AST in serum samples of treated groups differ significantly (p<0.05) when compared with negative control group (Group III). Treatment with methanol stem bark extract of Ficus platyphylla may have offered protection against CCl4 induced liver damage in experimental rats.

Protective Effect of Deer Skin Glue Polysaccharide on Alcoholic Liver Injury

Objective: To explore the protective effect of deer skin gum polysaccharide on acute alcoholic liver injury. Methods: The crude deer skin gum polysaccharide was extracted by extraction method, and purified by column chromatography to obtain pure deer skin gum polysaccharide, and its physical and chemical properties were identified by column chromatography, infrared spectroscopy and physical and chemical reaction experiments. The content of aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA) in mouse serum and the activity of GSH-Px, SOD and the content of MDA in the supernatant of liver homogenate were measured. The liver tissue sections of mice were observed to explore the protective effect of deer skin gum polysaccharide on acute alcoholic liver injury. Results: The deer skin gum polysaccharide was an acid polysaccharide with the pyranose structure. Its shape was white flocculent, with a molecular weight of 41.2 kDa and the purity of 86.71%. It had no starch, phenols, protein, amino acids and reducing sugar residues.The animal experiment results showed that compared with the model group, the deer skin gum polysaccharide middle and high dose groups could significantly increase the SOD and GSH-Px activities in the serum of mice (P<0.05), and reduce the ALT, AST activities and MDA content (P<0.05). At the same time, it could significantly increase the activity of SOD and GSH-Px in the supernatant of liver tissue homogenate (P<0.05), and significantly reduce the content of MDA (P<0.05). Conclusion: Deer skin gum polysaccharide has obvious protective effect on mice with acute alcoholic liver injury.

My career development with Ames test: A personal recollection

I first became acquainted with the Ames test at the very beginning of my career in 1978, when my task at the National Institute of Health Sciences (Tokyo) was to screen for mutagenicity of food additives used in Japan, using the Ames test. I also used this test to research the metabolic activation mechanisms of chemical carcinogens, in particular, the analgesic drug, phenacetin. This chemical was not mutagenic in Salmonella typhimurium TA100 with standard 9000 × g supernatant of liver homogenates (S9) from rat but was mutagenic with hamster S9. It was revealed that hamster S9 had much higher deacetylation activities than rat S9, which accounts for the species difference. Then, my work was focused on molecular biology. We cloned the genes encoding nitroreductase and acetyltransferase in Salmonella typhimurium TA1538. Plasmids carrying these genes made strain TA98 more sensitive to mutagenic nitroarenes and aromatic amines. Because of their high sensitivity, the resulting strains such as YG1021 and YG1024 are widely used to monitor mutagenic nitroarenes and aromatic amines in complex mixtures. Later, we disrupted the genes encoding DNA polymerases in TA1538 and classified chemical mutagens into four classes depending on their use of different DNA polymerases. I was also involved in the generation of gpt delta transgenic rodent gene mutation assays, which examine the results of the Ames test in vivo. I have unintentionally developed my career under the influence of Dr. Ames and I would like to acknowledge his remarkable achievements in the field of environmental mutagenesis and carcinogenesis.

Protective effect of raspberry extract on ConA-induced acute liver injuryin mice

In this experiment,the antioxidant capacity of raspberry extract and the protective effect on liver injury induced by ConA in mice were investigated. Balb/C male mice were randomly divided into six groups: normal group,model group,bicyclol control group( 200 mg·kg~(-1)),low-dose raspberry extract group( 200 mg·kg~(-1)),middle-dose raspberry extract group( 400 mg·kg~(-1)),and highdose raspberry extract group( 800 mg·kg~(-1)). Each group was intragastrically administered with drugs according to the body weight once a day. Seven days later,all of the groups except for the normal group were treated with ConA( 20 mg·kg~(-1)) through tail vein injection to establish the acute liver injury model. The mice were put to death 8 hours later. The organ indexes were calculated. These rum levels of ALT,AST and LDH and the activities of SOD,CAT,GSH and MDA in liver tissue were detected. HE staining was used to observe the pathological changes of liver tissue in mice. Western blot was used to detect the expressions of Bax,Bcl-2,Nrf2 and Keap-1. The antioxidant capacity of raspberry extract was measured by CAA assay. The results showed that,raspberry extract had a strong antioxidant capacity. Simultaneously,compared with the model group,raspberry extract can significantly improve the pathological conditions of liver,and significantly reduce ALT,AST and LDH activities in serum of liver injury mice( P<0. 01). The activities of SOD,CAT in liver homogenate supernatant were significantly increased in the high-dose group,the content of GSH increased,while the content of MDA was sharply declined in the high-dose group( P<0. 01). Meanwhile,raspberry extract down-regulated the expressions of Bax and Keap-1 and up-regulated the expressions of Bcl-2 and Nrf2. CAA showed that the compound raspberry extract had a strong antioxidant capacity. Therefore,raspberry extract has an obvious protective effect on acute liver injury induced by ConA in mice.

The processes and mechanism of antimony sequestered by red blood cells and its metabolic conjugation with hemoglobin in rats

Toxicity of antimony (Sb) to humans may occur through environmental, occupational, and therapeutic exposures. The underlying mechanism of its accumulation in red blood cells (RBCs) is not clear. Here, the processes and mechanism of RBCs sequestering Sb were explored through a series of in vitro and in vivo studies. These include binding affinity of Sb compounds with rat hemoglobin (Hb) and RBCs, acute exposure of antimony potassium tartrate (APTIII), and subchronic oral exposure of APTIII, potassium pyroantimonate (PPV) and antimony trioxide (ATIII). In vitro study indicated that parent form of Sb exhibited notable affinity with RBCs, while represented negligible affinity with Hb. However, after acute exposure to APTIII, over 93% of Sb in rat blood was integrated into Hb. Sb retaining in liver homogenate supernatants was substantially integrated into Hb, which indicated liver metabolism played a potential role influencing its ultimate partitioning in blood. Subchronic exposure of APTIII, PPV and ATIII to rats also demonstrated that most of Sb metabolites were integrated into Hb, regardless of which Sb compounds administered. Nano-HPLC–MS/MS analysis suggested that a dimethylated Sb species in pentavalent state [Sb(CH3)2O2H] was a major Sb group conjugated with Hb at Cys 104 and Cys 111 especially, in α chain of rat Hb. Hb-conjugated Sb did not affect Hb’s oxygen binding capability. As the deposit sites for both the parent forms and the metabolite, Sb re-released to circulation system due to RBCs and Hb degradation could have high substantial toxicological effects on its potential sites of action.

Determination of prodrug treosulfan and its biologically active monoepoxide in rat plasma, liver, lungs, kidneys, muscle, and brain by HPLC–ESI–MS/MS method

A prodrug treosulfan (TREO) is currently investigated in clinical trials for conditioning prior to hematopoietic stem cell transplantation. Bioanalysis of TREO and its active derivatives, monoepoxide (S,S-EBDM) and diepoxide, in plasma and urine underlay the pharmacokinetic studies of these compounds but cannot explain an organ pharmacological action or toxicity. Recently, distribution of TREO and S,S-EBDM into brain, cerebrospinal fluid, and aqueous humor of the eye has been investigated in animal models and the obtained results presented clinical relevance. In this paper, a selective and rapid HPLC–ESI–MS/MS method was elaborated and validated for the studies of disposition of TREO and S,S-EBDM in rat plasma, liver, lungs, kidneys, muscle, and brain. The two analytes and codeine, internal standard (IS), were isolated from 50μL of plasma and 100μL of supernatants of the tissues homogenates using ultrafiltration Amicon vials. Chromatographic resolution was accomplished on C18 column with isocratic elution. The limits of quantitation of TREO and S,S-EBDM in the studied matrices ranged from 0.11 to 0.93μM. The HPLC–MS/MS method was adequately precise and accurate within and between runs. The IS-normalized matrix effect differed among the tissues and was the most pronounced in a liver homogenate supernatant (approximately 0.55 for TREO and 0.35 for S,S-EBDM). Stability of the analytes in experimental samples was also established. The validated method for the first time enabled determination of TREO and S,S-EBDM in the six life-important tissues in rats following administration of the prodrug.

Zanthoxylum heitzii Modulates Ferric Nitrilotriacetate-Dependent Oxidative Alterations in Four Vital Organs: An In Vitro Organoprotective Model.

Ferric nitrilotriacetate (Fe-NTA) is a highly reactive compound used to induce degenerative disorders through oxidative stress (OS). Zanthoxylum heitzii (Z. heitzii) is a spice used as a medicinal plant to treat a variety of illnesses. This study investigated the ability of extracts from the leaves, fruits, roots, and barks of Z. Heitzii to inhibit Fe-NTA mediated oxidative damage in rats. The supernatant of rat liver homogenates was pretreated with the extracts for one hour before the induction of oxidative damage using a solution of Fe-NTA (400 mM). The activities of superoxide dismutase (SOD), catalase, and peroxidases were measured together with the marker of lipid peroxidation and the level of glutathione. The pretreated groups showed a significant increase in the activity of SOD, catalase, and peroxidases. The methanolic extract from the leaves of Z. heitzii (36.78 ± 3.30) and aqueous extract from the fruits (37.01 ± 2.52) showed the highest activities of SOD in the liver. The lowest concentration of MDA was found in the liver, and the glutathione was greater in the brain. Conclusively, these results suggest that Z. heitzii might be a chemoprotector which may be used in for prevention of distinct types of diseases induced by oxidative stress.

Effect of Microenvironment on Differentiation of Human Umbilical Cord Mesenchymal Stem Cells into Hepatocytes In Vitro and In Vivo.

Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) are considered to be an ideal cell source for cell therapy of many diseases. The aim of this study was to investigate the contribution of the microenvironment to the hepatic differentiation potential of hUCMSCs in vitro and in vivo and to explore their therapeutic use in acute liver injury in rats. We established a new model to simulate the liver tissue microenvironment in vivo using liver homogenate supernatant (LHS) in vitro. This induced environment could drive hUCMSCs to differentiate into hepatocyte-like cells within 7 days. The differentiated cells expressed hepatocyte-specific markers and demonstrated hepatocellular functions. We also injected hUCMSCs into rats with CCl4-induced acute hepatic injury. The hUCMSCs were detected in the livers of recipient rats and expressed the human hepatocyte-specific markers, suggesting that hUCMSCs could differentiate into hepatocyte-like cells in vivo in the liver tissue microenvironment. Levels of biochemistry markers improved significantly after transplantation of hUCMSCs compared with the nontransplantation group (P < 0.05). In conclusion, this study demonstrated that the liver tissue microenvironment may contribute to the differentiation of hUCMSCs into hepatocytes both in vitro and in vivo.

Hepatoprotective activity of the wild carrot chloroform-based fraction against CCl4-induced liver toxicity in mice

The liver plays a major role in detoxifying and clearing xenobiotics that may lead to liver damage and hepatic dysfunction. The present study was carried out to evaluate the hepatoprotective activities of the chloroform-based fraction of wild carrot (Daucus carota L. ssp. carota; Apiacae) on CCl4-induced liver damage in BALB/c mice. Liver damage was induced by intraperitoneal injection of CCl4 at a dose of 1 ml/kg body weight. Animals were treated with different doses of the extract (50, 100 and 200 mg/kg body weight). Serum and liver homogenate supernatants were analyzed for marker enzymes aspartate transaminase (AST), alanine transaminase (ALT), catalase (CAT), superoxide dismutase (SOD) and glutathione S-transferase (GST) activities. Pretreated groups with chloroform-based fraction reduced significantly the serum levels of AST and ALT. It also reversed the CCl4-induced decrease in SOD, CAT and GST levels thereby reducing significantly hepatic damage. HPLC analysis revealed that the chloroform-based fraction was rich in polyphenolic compounds such as luteolin, kaempferol, apigenin and quercetin, which could be partially responsible for the hepatoprotective effect. The current results suggest that chloroform-based fraction possess strong hepatoprotective activity against CCl4 induced liver damage at all doses used.

Impact of dietary oils and fats on lipid peroxidation in liver and blood of albino rats

ObjectiveTo investigate the effects of different dietary fat and oils (differing in their degree of saturation and unsaturation) on lipid peroxidation in liver and blood of rats. MethodsThe study was conducted on 50 albino rats that were randomly divided into 5 groups of 10 animals. The groups were fed on dietary butter (Group I), margarine (Group II), olive oil (Group III), sunflower oil (Group IV) and corn oil (Group V) for 7 weeks. After 12 h of diet removal, livers were excised and blood was collected to measure malondialdehyde (MDA) levels in the supernatant of liver homogenate and in blood. Blood superoxide dismutase activity (SOD), glutathione peroxidase activity (GPx), serum vitamin E and total antioxidant capacity (TAC) levels were also measured to determine the effects of fats and oils on lipid peroxidation. ResultsThe results indicated that no significant differences were observed in SOD activity, vitamin E and TAC levels between the five groups. However, there was significant decrease of GPx activity in groups IV and V when compared with other groups. The results indicated that feeding corn oil caused significant increases in liver and blood MDA levels as compared with other oils and fats. There were positive correlations between SOD and GPx, vitamin E and TAC as well as between GPx and TAC (r: 0.743; P<0.001) and between blood MDA and liver MDA (r: 0.897; P<0.001). The results showed also negative correlations between blood MDA on one hand and SOD, GPx, vitamin E and TAC on the other hand. ConclusionsThe results demonstrated that feeding oils rich in polyunsaturated fatty acids (PUFA) increases lipid peroxidation significantly and may raise the susceptibility of tissues to free radical oxidative damage.

Hepatoprotective activity of a new germanium-organic biologically active substance (medgerm) in experimental hepatitis

Aim. To study of the influence of new germanium-organic biologically active substance (medgerm) on lipid peroxidation and antioxidant system in rats with galactosamin-induced acute hepatitis. Methods. The experiments were carried out on Wistar male rats. Acute toxic hepatitis in rats was induced by intraperitoneal injection of D-galactosamine. Experimental animals were divided into 4 groups: first group (n=10) - intact animals that received 0.9% sodium chloride solution intraperitoneal injections throughout the observation period (control group); second group (n=40) - animals that received only D-galactosamine; third group (n=40) - rats that received medgerm intraperitoneal injections 7 days before and 7 days after the administration of D-galactosamine. The dose and administration regimen of medgerm were pre-defined. The fourth group (n=40) included rats that received Essentiale® N in the same mode as a comparator drug. Determination of thiobarbituric acid reactants, reduced glutathione levels in serum and liver homogenate supernatant, as well as superoxide dismutase and catalase activities were carried out in 1, 3, 5 and 7 days after the administration of liver toxins. Results. Medgerm has a significant effect on pro- and antioxidant homeostasis in acute toxic galactosamine-induced hepatitis. It effectively prevented the generation and accumulation of lipid peroxidation end products and preserved the activity of nonenzymatic and enzymatic antioxidant system parts. In animals treated with medgerm, studied parameters restored to reach the baseline level faster than in rats with galactosamine-induced hepatitis that were not treated with medgerm. Conclusion. The findings suggest that medgerm has the antioxidant activity and a membrane-mediated action in acute toxic hepatitis.

Influence of streptozocin-induced diabetes on reductive metabolism of acetohexamide in rat liver

Streptozocin-induced diabetes significantly decreased acetohexamide reductase activity of 10,000 g supernatant of liver homogenates from both male and female rats. However, the decrease in activity from female rats was smaller than that from male rats, thereby eliminating the sex difference in the activity of the 10,000 g supernatant. In male rats, the diabetes markedly decreased acetohexamide reductase activity only in the microsomal fraction of liver homogenate, whereas in female rats, it decreased the activity only in the cytosolic fraction. These results indicate that the mechanism for the decreasing effect of the diabetes on reductive metabolism of acetohexamide in 10,000 g supernatant differs between male and female rats.

Biotransformation of the topical glucocorticoids budesonide and beclomethasone 17 alpha,21-dipropionate in human liver and lung homogenate.

Tritiated budesonide and beclomethasone 17 alpha,21-dipropionate (BDP) were incubated with the 9000g supernatant of human liver homogenate. BDP was immediately hydrolysed to beclomethasone 17 alpha-propionate (BMP). BMP was then further biotransformed to polar metabolites. Budesonide was rapidly biotransformed (2-4 times more rapidly than BMP) to metabolites of low glucocorticoid potency. The compounds were also incubated with the 1000g supernatant of human lung homogenate. BDP was rapidly hydrolysed to BMP and then more slowly to beclomethasone. Budesonide was not biotransformed in the lung.

Promoting effect of the new chymotrypsin inhibitor FK-448 on the intestinal absorption of insulin in rats and dogs.

FK-448 is a potent and specific inhibitor of chymotrypsin, which enhances the intestinal absorption of insulin in rats and dogs resulting in a decrease in blood glucose levels in these animals. In dogs, the immunoreactive insulin (IRI) level of plasma rose proportionally to the decrease in blood glucose level. From in-vitro data, insulin was inactivated by pancreatic enzymes or the supernatants of intestine or liver homogenates. FK-448 suppressed the digestion of insulin by pancreatic enzymes and its enhancement of the intestinal absorption of insulin was found to be related to its inhibition of digestive enzymes, especially chymotrypsin.

Comparative bioactivation of the novel anti-tuberculosis agent PA-824 in Mycobacteria and a subcellular fraction of human liver

PA-824 is a 2-nitroimidazooxazine prodrug currently in Phase II clinical trial for tuberculosis therapy. It is bioactivated by a deazaflavin (F(420) )-dependent nitroreductase (Ddn) isolated from Mycobacterium tuberculosis to form a des-nitro metabolite. This releases toxic reactive nitrogen species which may be responsible for its anti-mycobacterial activity. There are no published reports of mammalian enzymes bioactivating this prodrug. We have investigated the metabolism of PA-824 following incubation with a subcellular fraction of human liver, in comparison with purified Ddn, M. tuberculosis and Mycobacterium smegmatis. PA-824 (250 µM) was incubated with the 9000 × g supernatant (S9) of human liver homogenates, purified Ddn, M. tuberculosis and M. smegmatis for metabolite identification by liquid chromatography mass spectrometry analysis. PA-824 was metabolized to seven products by Ddn and M. tuberculosis, with the major metabolite being the des-nitro product. Six of these products, but not the des-nitro metabolite, were also detected in M. smegmatis. In contrast, only four of these metabolites were observed in human liver S9; M3, a reduction product previously proposed as an intermediate in the Ddn-catalyzed des-nitrification and radiolytic reduction of PA-824; two unidentified metabolites, M1 and M4, which were products of M3; and a haem-catalyzed product of imidazole ring hydration (M2). PA-824 was metabolized by des-nitrification in Ddn and M. tuberculosis, but this does not occur in human liver S9 and M. smegmatis. Thus, PA-824 was selectively bioactivated in M. tuberculosis and there was no evidence for 'cross-activation' by human enzymes.

A pH/Enzyme-responsive tumor-specific delivery system for doxorubicin

To overcome the cardiotoxicity of doxorubicin, a self-assembled pH/enzyme-responsive system was developed. Cationic gelatin combined polyGC–DOX intercalation tightly to form compact nanoscale complexes (CPX1) which then combined by a pH-sensitive pegylated alginate to form CPX2. CPX2 could be digested and release DOX under the co-digestion of gelatinase (GA) and Dnase I when pH < 6.9. More importantly, tumor homogenate supernatant (THS) could effectively release DOX from CPX2 while the plasma and liver homogenate supernatant (LHS) could not, which was confirmed by an in vivo test. The results indicated that this formulation had the tumor-specific drug-release effect. This effect resulted in an increased drug concentration in tumor tissue and decreased content in heart and liver. The changed bio-distribution of DOX delivered by CPX2 greatly enhanced the anti-cancer activity and reduced the cardiotoxicity of the drug. The anti-cancer efficiency of DOX delivered by CPX2 is more than 2 times of the free doxorubicin, and the mortality caused by the high-dose DOX was completely prevented by CPX2. All results suggested that this easy-manufactured, cost-effective nanocomplex holds great promise to be developed into a formulation of doxorubicin and the other anthracyclines with high anti-cancer activity and low cardiotoxicity.

The Influence of Ethanol Pretreatment on the Effects of Nine Hepatotoxic Agents

The hepatotoxic effects of carbon tetrachloride (0.01 ml/kg i.p.), thioacetamide (50 mg/kg intraperitoneally), paracetamol (0.5 g/kg intraperitoneally), and allyl alcohol (0.05 ml/kg intraperitoneally) as estimated by determination of serum enzyme activities (GOT, GPT, SDH) were enhanced in mice treated with one oral dose of 4.8 g/kg ethanol 16 hrs. previously. Pretreatment of mice with ethanol did not increase the hepatotoxic actions of bromobenzene (0.25 ml/kg intraperitoneally), phalloidin (1.5 mg/kg intraperitoneally), alpha-amanitin (0.75 mg/kg intraperitoneally), and praseodymium (12 mg/kg intravenously) though there was a trend to higher enzyme activities in the case of bromobenzene. In guinea-pigs ethanol also aggravated CCl4-induced liver damage, but only strengthened the hepatotoxic activity of D-galactosamine (150 mg/kg intraperitoneally). Treatment with 4.8 g/kg ethanol did not influence liver glutathione levels in mice but increased aniline hydroxylation in the 9000 x g liver homogenate supernatant of mice and guinea-pigs. A dose of 2.4 g/kg ethanol, on the other hand, neither increased aniline hydroxylase activity nor enhanced carbon tetrachloride-induced hepatotoxicity in mice. It is assumed that the enhanced sensitivity to hepatotoxic agents after treatment with ethanol may be due to an enhanced microsomal activation of these substances.

Endogenous Uremic Substances are not Involved in the Reduced Hepatic Extraction of Metoprolol in Bilateral Ureter-Ligated Rats†

The hepatic extraction of metoprolol is reduced in rats with bilateral ureter ligation (BUL)-induced renal failure. The aim of the present study was to evaluate the effect of uremic substances on the hepatic metabolism of metoprolol in rats with BUL. The metabolic rate in the liver microsomes of BUL rats was similar to that in sham rats, and there was no significant difference between sham and BUL rats in the effect of the supernatant of liver homogenates on the metabolism. The rate of metabolism in the liver microsomes in the presence of the plasma of BUL rats was also similar to that in the presence of the plasma of sham rats. These findings indicated that uremic substances which accumulate in BUL rats do not directly inhibit the activity of CYP2D2, which is responsible for the metabolism of metoprolol in the rat liver.