Circular RNAs 0064286 and 0000475: Potential Diagnostic Biomarkers in Hepatocellular Carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC) is considered the highest recorded malignancy in Egypt. The shortage of appropriate biomarkers for early detection often results in the late diagnosis of the HCC. Circular RNAs (CircRNAs) are presented as long stranded non-coding RNA that combine covalently to make a sealed circular form which make them very stable. CircRNAs are known to have interpretative role in cancer development and metastasis. AIM: To examine the dysregulation of two new CircRNAs obtained from Circbase database (hsa_circ_0064286 and hsa_circ_0000475) in the serum of HCC patients as predictable diagnostic biomarkers of HCC and their correlation with some liver biochemical parameters. METHODS: Sixty clinically diagnosed HCC Egyptian patients and 25 healthy volunteers were enrolled in the study. Expression levels of the selected CircRNAs was evaluated in subjects' serum. Moreover, correlation with liver biochemical parameters, sensitivity, and specificity of studied CircRNAs were estimated. RESULTS: Both circular RNAs were significantly down regulated in HCC patients, which was negatively correlated with ALP, ALT, AST, AFP, and bilirubin levels. Circ_0064286 showed more sensitivity and specificity (88.3% and 96%, respectively). CONCLUSION: As far as we know, this is the first study that shed light on the expression levels of both circRNAs in Egyptian HCC patients. They may serve as potential biomarkers for HCC diagnosis. Moreover, those circRNAs draw attention as therapeutic targets for HCC through targeting their sponge miRNAs.

Effects of volatile oil constituents of Nigella sativa on carbon tetrachloride-induced hepatotoxicity in mice: evidence for antioxidant effects of thymoquinone.

Effects of the volatile oil constituents of Nigella sativa, namely, thymoquinone (TQ), p-cymene and alpha-pinene, on carbon tetrachloride (CCl4-indued acute liver injury were investigated in mice. A single dose of CCl4 (15 microl/Kg i.p.) induced hepatotoxicity 24 h after administration manifested biochemically as significant elevation of the enzymes activities of serum alanine transaminase (ALT, EC:2.6.1.2), asparate transaminase (AST, EC:2.6.1.1) and lactate dehydrogenase (LDH, EC: 1.1.1.27). The toxicity was further evidenced by a significant decrease of non-protein sulfhydryl(-SH) concentration, and a significant increase of lipid peroxidation measued as malondialdhyde (MDA) in the liver tissues. Administration of different doses of the TQ (4, 8, 12.5, 25 and 50 mg/Kg i.p.) did not alter the chosen biochemical parameters measured, while higher doses of TQ were lethal. The LD50 was 90.3 mg/Kg (77.9-104.7, 95% CL). Pretreatment of mice with different doses of TQ 1 h before CCl4 injection showed that the only dose of TQ that ameliorated hepatotoxicity of CCl4 was 12.5 mg/Kg i.p. as evidenced by the significant reduction of the elevated levels of serum enzymes as well as hepatic MDA content and significant increase of the hepatic nonprotein sulfhydryl(-SH) concentration. Treatment of mice with the other volatile oil constituents, p-cymene or alpha-pinene did not induce any changes in the serum ALT measured. In addition, i.p. administration of these compounds 1 h before CCl4 injection, did not protect mice against CC4-induced hepatotoxicity. The results of the present study indicate that TQ (12.5 mg/Kg, i.p.) may play an important role as antioxidant and may efficiently act as a protective agent against chemically-induced hepatic damage. In contrast, higher doses of TQ were found to induce oxidative stress leading to hepatic injury.

Thymoquinone protects against carbon tetrachloride hepatotoxicity in mice via an antioxidant mechanism.

Thymoquinone (TQ) is the major active component of the volatile oil of Nigella sativa seeds. The effects of TQ on carbon tetrachloride (CCl4)-induced hepatotoxicity was investigated in male Swiss albino mice. Carbon tetrachloride (20 microliters/Kg, i.p.) injected into mice, induced damage to liver cells and was followed by the increase in serum alanine aminotransferase (ALT) activity after 24 h. Oral administration of TQ in a single dose (100 mg/Kg) resulted in significant (p < 0.001) protection against the hepatotoxic effects of CCl4. TQ was tested as a substrate for mice hepatic DT-diaphorase in the presence of NADH. TQ appears to undergo reduction to dihydrothymoquinone (DHTQ). Reduction rates as a function of protein (liver homogenate) and substrate (TQ) concentrations are reported. An apparent K(m) of 0.1 mM and an apparent Vmax of 74 mumol/min/g liver were measured. TQ and DHTQ inhibited the in vitro non-enzymatic lipid peroxidation in liver homogenate (induced by Fe(3+)-ascorbate) in a dose dependent manner. In this in vitro model DHTQ was more potent in comparison with TQ and butylated hydroxytoluene (BHT). The IC50 for DHTQ, TQ and BHT were found to be 0.34, 0.87 and 0.58 microM respectively. The data suggest that the in vivo protective action of TQ against CCl4-induced hepatotoxicity may be mediated through the combined antioxidant properties of TQ and its metabolite DHTQ.

Effects of L-histidinol on the antitumour activity and acute cardiotoxicity of doxorubicin in mice.

L-Histidinol (LHL), a structural analogue of the essential amino acid l-histidine, can improve the therapeutic index of antimetabolites and alkylating agents. The objective of the study was to determine whether LHL would modulate the antitumour activity and acute cardiotoxicity of the anthracycline antibiotic, doxorubicin (DOX). LHL (1.0 mM) potentiated the cytotoxicity of DOX (0.05-0.8 microg ml-1) in cultured Ehrlich ascites carcinoma (EAC) cells. LHL (250 mg kg-1, i.p.) administered for five consecutive doses at 2-h intervals starting 2 h before DOX (5 mg kg-1, i.p.) single injection, enhanced the antitumour activity of DOX in EAC-bearing mice as manifested by a significant increase in average life span and cure rate of mice. In normal mice, LHL, in the same dose regimen, could not alter the acute cardiotoxicity and lethality of DOX (10 mg kg-1, i.p.). The present data indicate that LHL may improve the therapeutic efficacy of DOX in EAC-bearing mice without compromising its toxicity. Also, our finding supports the LHL/anticancer drug combination approach.

Vitamin E protects the brain against oxidative injury stimulated by excessive aluminum intake.

The effect of feeding groups of mice with a diet containing 2000, 4000 and 6000 micrograms aluminum (Al3-/g) for two weeks (subacute) or 2000 and 4000 micrograms Al3+/g for eight weeks (subchronic) as well as the coadministration of vitamin E (alpha-tocopherol) 500 micrograms/g with Al3+, on the status of glutathione (GSH) and lipid peroxides as thiobarbituric acid reactive substances (TBARS) in whole brain tissues were evaluated. Changes in TBARS were further evaluated in vitro following the incubation of brain homogenates of the Al(3+)-fed mice in the presence of 50 microM FeSO4. The results of subacute experiments revealed that the brain levels of GSH were significantly decreased only in the group of mice that received 6000 micrograms Al3+/g diet (P < 0.05) and this effect was partially ameliorated when vitamin E was coadministered with Al3+. TBARS were significantly increased in vitro only in the presence of free iron ions and depended on the concentration of Al3+ in the diet. The effect was opposed by the vitamin E intake. Following subchronic Al3+ intake, the GSH content of the brain was significantly decreased only in the group of mice that received 4000 micrograms Al3+/g diet (P < 0.01), while TBARS were significantly increased in the brain tissues in vivo as well as in the presence of free iron ions in vitro. However, coadministration of vitamin E with Al3+ for eight weeks preserved GSH levels and decreased TBARS in the brain of mice in vivo and in the presence of free iron ions in vitro. It is concluded that the long term administration of vitamin E may prevent Al3(+)-stimulated oxidative injury in the brain.

Effect of L-histidinol on cisplatin nephrotoxicity in the rat.

The effect of L-histidinol (LHL) on the acute nephrotoxicity produced by cisplatin (CDDP; 6 mg/kg, i.v.) was investigated in the rat. Intraperitoneal administration of LHL (100 mg/kg x 5 doses, 2 h apart) starting 2 h prior to CDDP single injection produced significant protection of renal function. The attenuation of nephrotoxicity was evidenced by significant reductions in serum urea and creatinine concentrations, decreased polyuria, reduction in body weight loss, marked reduction in urinary fractional sodium excretion and glutathione-S-transferase (GST) activity, and increased urine/serum creatinine ratio as well as increased creatinine clearance. LHL significantly ameliorated the toxic renal biochemical changes induced by CDDP. Renal lipid peroxides, glutathione levels and GST activity showed a marked tendency towards the normal values. Accumulation of platinum in renal tissues was significantly decreased in the presence of LHL. It is concluded that LHL can act as a nephroprotectant, and it is suggested that it would have beneficial effects on the kidney in clinical settings.

Thymoquinone ameliorates the nephrotoxicity induced by cisplatin in rodents and potentiates its antitumor activity.

The effects of thymoquinone (TQ) on cisplatin-induced nephrotoxicity in mice and rats were studied. Oral administration of TQ (50 mg/L in drinking water) for 5 days before and 5 days after single injections of cisplatin (5 mg/kg, i.v., in rats and 7 or 14 mg/kg, i.p., in mice) greatly ameliorated cisplatin-induced nephrotoxicity in both species. In rats, i.v. cisplatin caused 4- and 5-fold elevations in serum urea and creatinine, a 235% increase in urine volume, a 41% increase in kidney weight, 8.5-fold decrease in creatinine clearance, and extensive histological damage 5 days after treatment. In mice, similar alterations in kidney function were observed. TQ-induced amelioration of cisplatin nephrotoxicity was evident by significant reductions in serum urea and creatinine and significant improvement in polyuria, kidney weight, and creatinine clearance. The protective effects of TQ against cisplatin-induced nephrotoxicity in the rat were further confirmed by histopathological examination. To evaluate the possible modification of the antitumor activity of cisplatin by TQ, we studied their interaction in Ehrlich ascites carcinoma (EAC) bearing mice. The results revealed that TQ potentiated the antitumor activity of cisplatin. The current study suggests that TQ may improve the therapeutic index of cisplatin.

Vitamin E protects against bacterial endotoxin-induced increase of plasma corticosterone and brain glutamate in the rat.

Plasma corticosterone (CS) and brain free aminoacids were determined in male rats 2 hr after acute exposure to bacterial endotoxin stress BES (2.0 mg/kg i.p. of lipopolysaccharide, LPS). A significant increase in the levels of plasma CS and brain taurine (Tau), aspartate (As), glutamate (Glu), glycine (Gly) and valine (Val) was observed following BES. When vitamin E (alpha-tocopherol acetate AT) was given orally (0.25 gm/kg/day) 4 days before induction of BES, the plasma CS as well as the brain Glu levels were significantly reduced to the control values. These results indicate that plasma CS and brain Glu may be involved in the mechanisms by which AT protects against the neurotoxicity of BES.

Spectrophotometric assay for superoxide dismutase based on the nitroblue tetrazolium reduction by glucose-glucose oxidase.

A new spectrophotometric assay of superoxide dismutase (SOD) is described. The assay is based on the SOD-mediated inhibition in the rate of nitroblue tetrazolium reduction to the blue formazan at alkaline pH. The optimized assay of SOD is performed in 50 mM glycine-NaOH buffer, pH 9.5, at 25 degrees C. The SOD concentration is determined from the V/v ratio of rates measured in the absence (V) or the presence (v) of SOD. One unit of SOD has been defined as the concentration that decrease the rate to 50% (V/v = 2). The assay is simple, sensitive, uses commercially available reagents, rapid and easy to perform and could be used routinely for monitoring superoxide dismutase levels in purified protein fractions.

Evidence for superoxide radical production by a simple flavoprotein: glucose oxidase.

Nitroblue tetrazolium was reduced to blue formazan during the oxidation of glucose by glucose oxidase. The rate of blue color formation was dependent on the concentrations of glucose, nitroblue tetrazolium and glucose oxidase. The rate of the reaction was negligible below pH 8.4, but sharply increased with increasing pH. The reduction of nitroblue tetrazolium was inhibited by superoxide dismutase, consistent with the participation of superoxide anion radical in the reaction.

Changes in free amino acids in peripheral blood (PB) lymphocytes and polymorphonuclear (PMN) leukocytes after treatment with diazepam.

The effect of single and chronic (15 days) i.p. injections (1.0 and 8.0 mg/kg) of diazepam (DZ) on free amino acid profile in peripheral blood (PB) lymphocytes and polymorphonuclear (PMN) leukocytes of male Wistar Albino rats were investigated. Depletion of some free amino acids was observed in the lymphocytes (mixed T- and B-lymphocytes) and PMN leukocytes (91-95%) neutrophils especially after chronic DZ-treatment. A dose-dependent depletion in the lymphocyte amino acids, Tau, Gly, Ala, Met and Ile, was found after both acute and chronic DZ-treatment. A similar depletion of Tau, Asp, Glu and Met appeared in the PMN leukocytes after single doses as well as chronic DZ-treatment. These results suggest that administration of 1.0-8.0 mg/kg of DZ in single dose or after chronic administration may interfere with the transport of certain important amino acids and/or protein turnover in PB lymphocytes and PMN leukocytes. On the other hand, the basic amino acids Lys, His and Arg were significantly increased in PMN leukocytes after chronic administration of 1.0 mg/kg DZ. It was suggested that the increased levels of the basic amino acids in the neutrophils may interact with the intracellular changes in pH that normally accompany the respiratory burst.