The correlation between renal function and bone mineral density.

AIM: The moderate, severe chronic kidney disease (CKD) and end stage renal disease (ESRD) are well-recognized risk factors of bone loss. However, it is uncertain whether mild CKD stage affects bone mineral density (BMD). The objective of this study is to investigate whether mild and/or more severe reduction of GFR is associated with BMD decreasing. METHODS: Between April and November 2011, 305 patients were included in this study. Estimated glomerular filtration rate (GFR) was calculated by Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. According to the GFR, we divided the participants into 3 groups: CKD stage I, stage II and stage ≥III. BMD was compared between different groups. RESULTS: Eighty-one women and 65 men were enrolled in the final study. The average age was 65.6. According to the eGFR, 54 (37.0%), 70 (47.9%) and 22 (15.1%) participants were classified into CKD stage I, II and ≥III, respectively. The mean T-score was -1.351±1.879.A significant bone loss was found in the stage ≥III group, especially in women (P<0.05). BMD decreasing was found significantly in male participants with CKD stage II (P=0.041). CONCLUSION: BMD significantly decreased in men with mild renal dysfunction. A significant BMD decreasing was also found in the group of CKD stage ≥III, especially in women. We suggest osteoporosis screening is necessary in patients with poor renal function.

Berberine induces apoptosis through a mitochondria/caspases pathway in human hepatoma cells.

Berberine, a main component of Coptidis Rhizoma, is a plant alkaloid with a long history of medicinal use in Chinese medicine. Berberine has indicated significant antimicrobial activity against a variety of organisms including bacteria, viruses, fungi. The mechanism by which berberine initiates apoptosis remains poorly understood. In the present study, we demonstrated that berberine exhibited significant cytotoxicity in hepatoma HepG2 cells but is ineffective in Chang liver cells. Herein we investigated cytotoxicity mechanism of berberine in HepG2 cells. The results showed that HepG2 cells underwent internucleosomal DNA fragmentation after 24-h treatment with berberine (50 microM). Moreover, berberine induced the activation of caspase-8 and -3, and caused the cleavage of poly ADP-ribose polymerase (PARP) and the cytochrome c release, whereas the expression of Bid and anti-apoptosis factor Bcl-XL were decreased markedly. The loss of mitochondrial membrane potential (Delta psim) at 24 h and activation of Fas at 12 h were also seen in the berberine-treated HepG2 cells. These findings supported the fact that the inhibitors of caspases, DEVD-FMK, IETD-FMK and VAD-FMK, prevented apoptosis and restored the expression of Bcl-XL, Bcl-2 and Bid. These results indicated that the potential of anti-hepatoma activity of berberine may be mediated through a caspases-mitochondria-dependent pathway.

Hibiscus protocatechuic acid inhibits lipopolysaccharide-induced rat hepatic damage.

Hibiscus protocatechuic acid (PCA), a phenolic compound found in the dried flowers of Hibiscus sabdariffa L. (Malvaceae), was demonstrated to have an antioxidant effect in vitro and in vivo, and an antitumor property in our previous study. In the present study, we used lipopolysaccharide (LPS, an endotoxin) to induce rat liver inducible nitric oxide synthase (iNOS), and found that pretreatment with PCA decreased the liver iNOS and the serum total nitrite induced by LPS. Our investigation showed that pretreatment of rats with PCA (0.2 and 0.5 mmol/kg dosed by gavage) for 5 days significantly decreased the serum levels of the hepatic enzyme markers alanine- and aspartate aminotransferase (ALT, alanine aminotransferase; AST, aspartate aminotransferase) induced by the 6-h treatment with LPS (i.p.; 5 mg/kg). Histopathological evaluation of the rat livers revealed that PCA reduced the incidence of liver lesions induced by LPS, including neutrophil infiltration, congestion, and liver cell swelling induced by LPS in rats. We conclude that PCA, an antioxidant, presents an inhibitory potential on iNOS and hepatic damage induced by LPS.

Tumor-promoting effect of GGN-MRP extract from the Maillard reaction products of glucose and glycine in the presence of sodium nitrite in C3H10T1/2 cells.

GGN-MRP is an extract from the Maillard reaction products of nitrite with glucose and glycine in the Maillard browning system. No genotoxicity of GGN-MRP in culture hepatocyte was found. A two-stage transformation protocol was used to transform chemically mouse embryo fibroblast C3H10T1/2 cells. To initiate transformation, the cells were treated with benzo[a]pyrene [B(a)P; 0.1 microg/mL], and GGN-MRP (0.01, 0.1, and 1.0 mg/mL) was employed to subsequently complete the transformation process. Malignant transformed foci were formed in B(a)P-initiated and GGN-MRP-promoted C3H10T1/2 cells after 8 weeks. Cells treated with GGN-MRP alone failed to induce transformation. However, cells initiated with B(a)P and promoted by GGN-MRP demonstrated oncogenic properties. Transformed colonies derived from GGN-MRP-treated cells exhibited enhanced growth rate, anchorage independence, and tumorgenicity in animals relative to parent cells. These results indicated that GGN-MRP contains a tumor promoter and may induce tumor promotion by two-stage oncogenesis.

Synergistic effects of nicotine on arecoline-induced cytotoxicity in human buccal mucosal fibroblasts.

Areca quid chewing has been linked to oral submucous fibrosis and oral cancer. Arecoline, a major areca nut alkaloid, is considered to be the most important etiologic factor in the areca nut. In order to elucidate the pathobiological effects of arecoline, cytotoxicity assays, cellular glutathione S-transferase (GST) activity and lipid peroxidation assay were employed to investigate cultured human buccal mucosal fibroblasts. To date, there is a large proportion of areca quid chewers who are also smokers. Furthermore, nicotine, the major product of cigarette smoking, was added to test how it modulated the cytotoxicity of arecoline. At a concentration higher than 50 microg/ml, arecoline was shown to be cytotoxic to human buccal fibroblasts in a dose-dependent manner by the alamar blue dye colorimetric assay (P<0.05). In addition, arecoline significantly decreased GST activity in a dose-dependent manner (P<0.05). At concentrations of 100 microg/ml and 400 microg/ml, arecoline reduced GST activity about 21% and 46%, respectively, during a 24 h incubation period. However, arecoline at any test dose did not increase lipid peroxidation in the present human buccal fibroblast test system. The addition of extracellular nicotine acted synergistically on the arecoline-induced cytotoxicity. Arecoline at a concentration of 50 microg/ml caused about 30% of cell death over the 24 h incubation period. However, 2.5 mM nicotine enhanced the cytotoxic response and caused about 50% of cell death on 50 microg/ml arecoline-induced cytotoxicity. Taken together, arecoline may render human buccal mucosal fibroblasts more vulnerable to other reactive agents in cigarettes via GST reduction. The compounds of tobacco products may act synergistically in the pathogenesis of oral mucosal lesions in areca quid chewers. The data presented here may partly explain why patients who combined the habits of areca quid chewing and cigarette smoking are at greater risk of contracting oral cancer.

Induction of apoptosis by esculetin in human leukemia cells.

Esculetin, a coumarin compound, has been shown to exhibit antioxidant and anti-inflammatory effects. In the present study, esculetin was found to inhibit the survival of human promyelocytic leukemia HL-60 cells in a concentration-dependent and time-dependent manner. HL-60 cells underwent internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis after a 24-h treatment with esculetin (100 microM). Flow cytometric analysis showed that the hypodiploid nuclei of HL-60 cells were increased to 40.93% after a 36-h treatment with esculetin (100 microM). Further investigation showed that esculetin induced the release of cytochrome c from mitochondria into cytosol in a time-dependent and concentration-dependent manner. Moreover, esculetin application reduced Bcl-2 protein expression to 58% after 9 h as compared with that time at 0. Cysteine protease 32 kDa proenzyme (CPP32), a caspase 3, was activated and its substrate, poly (adenosine diphosphate-ribose) polymerase, was cleaved after a 24-h treatment of HL-60 cells with esculetin. These data suggest that esculetin induces apoptosis in human leukemia cells by increasing cytosolic translocation of cytochrome c and activation of CPP32.

Gaseous nitric oxide-induced 8-nitroguanine formation in human lung fibroblast cells and cell-free DNA.

A time- and dose-dependent increase in 8-nitroguanine (8-NO(2)-G) was observed in human lung fibroblast cells (MRC-5) after treatment with gaseous NO-saturated buffer. It was also found that treatment with the inhibitor of inducible nitric oxide synthase (iNOS), N(G)-nitro-l-arginine methyl ester, significantly reduced the 8-NO(2)-G level in the gaseous NO-saturated buffer-treated MRC-5 cells. These results provide evidence indicating that NO gas causes DNA damage in mammalian cells, which involves the activation of iNOS and the subsequent generation of endogenous NO. On the other hand, a time- and dose-dependent increase in 8-NO(2)-G was also observed while DNA (isolated from MRC-5 cells) was incubated with gaseous NO-saturated buffer. These results suggest that part of the 8-NO(2)-G formation was due to direct modification of gaseous NO on DNA. Furthermore, an increase in nitrite concentration was found in both cell-free and MRC-5 cell-conditioned medium treated with gaseous NO-saturated buffer. Collectively, gaseous NO induced DNA damage by forming 8-NO(2)-G, a modification performed directly by the treated gaseous NO and indirectly by the following induction of endogenous NO. This effect might be an important pathway in genotoxicity of nitric oxides, and 8-NO(2)-G could act as a specific marker for DNA damage induced by gaseous NO, a common contaminatant in air pollution and cigarette smoke.

Cytotoxicity effects of di- and tri-hydroxybenzaldehydes as a chemopreventive potential agent on tumor cells.

As part of our earlier search for new compounds with improved biological activities including antioxidant, anti-inflammatory, and tumor growth inhibition activities, we synthesized 2,4,5-trihydroxybenzaldehyde (2,4,5-THBA) from commercially available Sesamol. First we examined the free radical-quenching capacity of 2,4,5-THBA, 3,4-dihydroxybenzaldehyde (3,4-DHBA), and 2,5-dihydroxybenzaldehyde (2,5-DHBA) in vitro by 1,1-diphenyl-1,2-picryhydrazylradical (DPPH) test. The antioxidant bioactivity was also evaluated using the model of t-butyl hydroperoxide (t-BHP)-induced cytotoxicity in rat primary hepatocytes. Finally, three chemicals were tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay on human nasopharynx carcinoma cells (KB cells), human hepatoblastoma (HepG2), and human leukemia HL-60 cells. The 2,4,5-THBA shows significant cytotoxicity on HL-60 cells. The results suggest that 2,4,5-THBA may be a potential chemopreventor or chemotherapy agent against HL-60 cells.

Inhibitory effect of esculetin on oxidative damage induced by t-butyl hydroperoxide in rat liver.

Increasing evidence regarding free radical-generating agents and inflammatory processes suggests that accumulation of reactive oxygen species can cause hepatotoxicity. A short-chain analog of lipid hydroperoxide, t-butyl hydroperoxide (t-BHP), can be metabolized to free radical intermediates by cytochrome P-450 in hepatocytes, which in turn can initiate lipid peroxidation, affect cell integrity and result in cell injury. In this study, we used t-BHP to induce hepatotoxicity in vitro and in vivo and determined the antioxidative bioactivity of esculetin, a coumarin compound. Our investigations showed that pretreatment with esculetin (5-20 microg/ml) significantly decreased the leakage of lactate dehydrogenase (LDH) and alanine transaminase (ALT), and also decreased the formation of malondialdehyde (MDA) in primary cultured rat hepatocytes induced by a 30-min treatment with t-BHP. An in vivo study in rats showed that pretreatment with esculetin (i.p.) at concentrations of 0.5 and 5 mg/kg for 5 days before a single i.p. dose of t-BHP (0.1 mmol/kg) significantly lowered the serum levels of the hepatic enzyme markers (ALT and AST) and reduced oxidative stress in the liver. Histopathological evaluation of the rat livers revealed that esculetin reduced the incidence of liver lesions induced by t-BHP, including hepatocyte swelling, leukocyte infiltration, and necrosis. Based on the results described above, we speculate that esculetin may play a chemopreventive role via reducing oxidative stress in living systems.

Tumor promotion of N-nitroso-N-(3-keto-1, 2-butanediol)-3'-nitrotyramine derived from nitrosation of Maillard reaction product in CD-1 mice.

N-Nitroso-N-(3-keto-1,2-butanediol)-3'-nitrotyramine (NO-NTA) is a product of a model browning system generated in the presence of sodium nitrite. Our previous study showed that NO-NTA had genotoxicity and proved to be an initiator and promoter on mouse C3H10T1/2 cells. In this study, a two-stage skin carcinogenesis protocol was used to promote CD-1 mouse skin carcinogenesis using NO-NTA. Twice weekly, for 38 weeks, topical application of NO-NTA at the concentration of 250 nmol to mice previously initiated with benzo(a)pyrene (BaP) caused 90% tumor incidence. However, no tumors were observed in mice treated with BaP or treated with NO-NTA alone. The NO-NTA-promoted tumors that were observed histologically in mice showed well-differentiated squamous cell carcinoma with invasion into the subcutaneous region. Application of the same amount of NO-NTA not only caused significant induction of hyperplasia but also epidermal ornithine decarboxylase (ODC) activity. Treatment of mouse skin (1 cm(2)) with various amounts of NO-NTA (10, 50, or 250 nmol) caused production of hydrogen peroxide by 1.63-, 1.91-, and 2. 38-fold, respectively, and marked induction of myeloperoxidase (MPO) by 21-, 39-, and 61-fold. These results indicate that NO-NTA is a new tumor promoter and may induce tumor promotion by oxidant stress in CD-1 mouse skin.

Induction of apoptosis by hibiscus protocatechuic acid in human leukemia cells via reduction of retinoblastoma (RB) phosphorylation and Bcl-2 expression.

Hibiscus protocatechuic acid (PCA), a phenolic compound isolated from the dried flower of Hibiscus sabdariffa L. (Malvaceae), demonstrated antioxidant and antitumor promotion effects in our previous study. In the present study, Hibiscus PCA was found to inhibit the survival of human promyelocytic leukemia HL-60 cells in a concentration- and time-dependent manner. The study revealed that HL-60 cells underwent internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis after a 9-hr treatment with Hibiscus PCA (2 mM). Flow cytometric analysis of the DNA content of cells treated with PCA for 12 hr showed that the cells were distributed mainly in the hypodiploid phase (apoptotic peak, 46.7%), less in the G(1) (34.2%) and S phase (14.0%), and few in the G(2)/M phase (5.1%). Moreover, PCA treatment caused an increase in the level of hypophosphorylated retinoblastoma (RB; 180% of control at the 6-hr time point) and, on the contrary, a decline in hyperphosphorylated RB. A rapid loss of RB was observed when the treatment period was extended. Further studies showed that Hibiscus PCA application reduced Bcl-2 protein expression to 47%, and increased Bax protein expression to 181% after 1.5 hr as compared with time 0. Overexpression of Bcl-2 in HL-60 cells delayed the occurrence of Hibiscus PCA-induced apoptosis. These data suggest that Hibiscus PCA is an apoptosis inducer in human leukemia cells, and that RB phosphorylation and Bcl-2 protein may play a crucial role in the early stage.

Protective effect of Hibiscus anthocyanins against tert-butyl hydroperoxide-induced hepatic toxicity in rats.

Hibiscus anthocyanins (HAs), a group of natural pigments occurring in the dried flowers of Hibiscus sabdariffa L., which is a local soft drink material and medical herb, were studied for antioxidant bioactivity. The preliminary study showed that HAs were able to quench the free radicals of 1,1-diphenyl-2-picrylhydrazyl. This antioxidant bioactivitiy was further evaluated using the model of tert-butyl hydroperoxide (t-BHP)-induced cytotoxicity in rat primary hepatocytes and hepatotoxicity in rats. The results demonstrated that HAs, at the concentrations of 0.10 and 0.20 mg/ml, significantly decreased the leakage of lactate dehydrogenase and the formation of malondialdehyde induced by a 30-min treatment of t-BHP (1.5 mM). The in vivo investigation showed that the oral pretreatment of HAs (100 and 200 mg/kg) for 5 days before a single dose of t-BHP (0.2 mmol/kg, ip) significantly lowered the serum levels of hepatic enzyme markers (alanine and aspartate aminotransferase) and reduced oxidative liver damage. The histopathological evaluation of the liver revealed that Hibiscus pigments reduced the incidence of liver lesions including inflammatory, leucocyte infiltration, and necrosis induced by t-BHP in rats. Based on the results described above, we speculate that Hibiscus pigments may play a role in the prevention of oxidative damage in living systems.

Synergistic effects of peroxynitrite on arecoline-induced cytotoxicity in human buccal mucosal fibroblasts.

Epidemiological studies have demonstrated a clear association between betel nut chewing and an increased risk for oral mucosal lesions. Arecoline, the most abundant betel alkaloid, is considered the most important etiologic factor in betel nuts. In addition, most betel nut chewers are also smokers. In order to elucidate the potential toxicological implications of interactions of arecoline and peroxynitrite (a reaction product of cigarette smoking), cell viability, and cellular levels of glutathione (GSH) were investigated, using cultured human buccal mucosal fibroblasts. At a concentration higher than 0.8 mM, arecoline was cytotoxic to buccal mucosal fibroblasts in a concentration- and time-dependent manner. Arecoline also depleted intracellular GSH in a dose-dependent manner (P<0.05). The addition of extracellular peroxynitrite acted as a synergistic effect on the arecoline-induced cytotoxicity (P<0.05). Furthermore, at a concentration of 0.8 mM, arecoline depleted intracellular GSH by about 42%, while 2 mM peroxynitrite enhanced the arecoline-depleted GSH level further to 86% as compared with the control. During GSH depletion, arecoline may render the human buccal mucosal fibroblasts more vulnerable to other reactive agents within cigarette smoking. Taken together, we suggest that people who combine the habits of betel nut chewing with cigarette smoking could be more susceptible to oral mucosal damage than betel quid chewing alone.

Inhibitory effect of 12-O-tetradecanoylphorbol-13-acetate-caused tumor promotion in benzo[a]pyrene-initiated CD-1 mouse skin by baicalein.

The effects of topical application of baicalein on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced promotion of skin tumors, hyperplasia, ornithine decarboxylase activity, and inflammation were evaluated in female CD-1 mice. Topical application of baicalein (0.08, 0.16, or 0.2 mumol) with TPA (5 nmol) twice weekly for 24 weeks to mice previously initiated with benzo[a]pyrene inhibited the number of TPA-induced tumors per mouse significantly. Preapplication of the same amount of baicalein also afforded significant protection against TPA-induced hyperplasia in the ear skin. Topical application of baicalein inhibited tumor promoter-caused induction of epidermal ornithine decarboxylase activity by TPA (5 nmol). The topical application of baicalein (0.008, 0.016, or 0.02 mumol) inhibited TPA-induced edema of mouse ears by 88%, 96%, or 97%, respectively. Pretreatment of mouse skin with various amounts of baicalein caused inhibition of H2O2 and myeloperoxidase formation by TPA. These results indicate that baicalein can be a potential cancer-chemopreventive agent against tumor promotion.

Protective effects of capillarisin on tert-butylhydroperoxide-induced oxidative damage in rat primary hepatocytes.

Capillarisin (Cap), a main constituent of Artemisia capillaris (Compositae), was studied for its antioxidant bioactivity. In the preliminary study, Cap expressed a antioxidant property by its capacity for quenching the free radicals of 1,1-diphenyl-2-picrylhydrazyl (DPPH). This antioxidant bioactivity of Cap was investigated further using a model of t-butylhydroperoxide (t-BHP)-induced cytotoxicity and genotoxicity in rat primary hepatocytes. Results presented here demonstrate that Cap, at concentrations of 0.01-1.00 mg/ml, significantly decreased the leakage of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) and the formation of malondialdehyde (MDA) induced by 30 min treatment of t-BHP (1.5 mM) in primary cultured rat hepatocytes. Cap also attenuated the t-BHP-induced diminution of glutathione (GSH) and high level of DNA repaired synthesis. These results lead to speculation that Cap presents inhibitory effects against t-BHP-caused cytotoxicity and genotoxicity in rat primary hepatocyte cultures at least via two distinct pathways, stabilizing the GSH system and quenching free radicals.

Tumor promoting effect of N-nitroso-N-(2-hexanonyl)-3'-nitrotyramine (a nitrosated Maillard reaction product) in benzo(a)pyrene-initiated mouse skin carcinogenesis.

N-nitroso-N-(2-hexanonyl)-3'-nitrotyramine (NO-HNTA) is a product generated in a model browning system in the presence of sodium nitrite. The chemical structure of this compound has been confirmed by UV, mass, nuclear magnetic resonance and infrared spectroscopy in our study. Twenty weeks, twice weekly, topical application of NO-HNTA at the concentration of 10, 50 and 250 mumol to mice previously initiated with benzo(a)pyrene (B[a]P) increased their tumor formation by 3.2-, 4.6- and 5.8-fold respectively. Application of the same amount of NO-HNTA not only caused significant induction of hyperplasia but also the activity of epidermal ornithine decarboxylase (ODC). Treatment of mouse skin with various amounts of NO-HNTA (10, 50 and 250 mumol) caused production of hydrogen peroxide by 1.38-, 1.95- and 3.26-fold respectively, and induction myeloperoxidase (MPO) by 24-, 63- and 102-fold. These results indicate that the formation of NO-HNTA or its derivatives derived from the reaction of tyrosine and glucose in the presence of sodium nitrite has the potential as a tumor promoter.

Inhibitory effect of Hibiscus protocatechuic acid on tumor promotion in mouse skin.

Hibiscus protocatechuic acid (PCA), a phenolic acid isolated from Hibiscus sabdariffa L., was evaluated for its ability to inhibit the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced promotion in skin tumors of female CD-1 mice. Topical application of PCA (5, 10 or 20 micromol) 5 min prior to TPA (15 nmol) treatment twice weekly for 20 weeks to mice which were initiated with benzo[a]pyrene (B[a]P) inhibited the incidence of tumors in mice to 81.3, 62.5 and 56.3%, respectively, while all mice in the TPA-treated group developed tumors. The average number of tumors in mice pretreated with PCA was 2-4 and that of mice treated only with TPA was 6.6. The protection effects of PCA were also presented by its significant suppression on the TPA-induced hyperplasia in the skin and edema of mouse ears by 65 and 73% at doses of 10 and 20 micromol, respectively. When it was applied to the dorsal surface of CD-1 mice before TPA application, PCA (5, 10 or 20 micromol) inhibited the induction of epidermal ornithine decarboxylase (ODC) activity by 5 nmol TPA and myeloperoxidase (MPO) activity by 6.5 nmol TPA. The same doses of PCA also reduced the formation of hydrogen peroxide in the mouse skin to an inhibition of 61, 84 and 89%, respectively, when compared with that of the TPA-treated group. These results indicate that PCA possesses potential as a cancer chemopreventive agent against tumor promotion.

Protective effects of dried flower extracts of Hibiscus sabdariffa L. against oxidative stress in rat primary hepatocytes.

Dried flower extracts of Hibiscus sabdariffa L., a local soft drink material and medical herb, was found to possess antioxidant activity in the present study. In the preliminary studies, antioxidant potential of three fractions of the ethanol crude extract (HS-C: chloroform-soluble fraction; HS-E: ethyl acetate soluble fraction; HS-R: residual fraction) obtained from the dried flowers of Hibiscus sabdariffa L. were evaluated by their capacity of quenching 1,1 -diphenyl-2-picrylhydrazyl (DPPH) free radical and inhibiting xanthine oxidase (XO) activity. HS-E showed the greatest capacity of scavenging free radical (EC50=0.017mg/ml), and HS-C showed the strongest inhibitory effect on XO activity (EC5o=0.742 mg/ml). Furthermore, antioxidant bioactivities of these crude extracts were investigated using a model of tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in rat primary hepatocytes. All fractions were found to inhibit significantly the unscheduled DNA synthesis (UDS) induced by t-BHP at a concentration of 0.20 mg/ml. HS-C and HS-E also decreased the leakage of lactate dehydrogenase (LDH) and the formation of malondialdehyde (MDA) induced by t-BHP (1.5 mM) considerably at a concentration of 0.10 and 0.20 mg/ml in the rat primary hepatocyte cultures. These results indicated that the dried flower extracts (HS-C and HS-E) of H. sabdariffa L. protect rat hepatocytes from t-BHP-induced cytotoxicity and genotoxicity by different mechanisms.

Hepatotoxicity of 1,3,5-trinitro-2-acetyl pyrrole derived from nitrosation of Maillard reaction product in BALB/C mouse.

1,3,5-Trinitro-2-acetyl pyrrole (TNAP) is a product derived from the reaction of 2-acetyl pyrrole with nitrite in the model of Maillard browning systems. This compound is moderately mutagenic to the Salmonella strains TA98 and TA100 and is markedly cytotoxic to mouse C3H10T1/2 cells. Experiments are performed to investigate the effects of TNAP on the hepatic toxicity in mouse. Male BALB/C mice were subjected to a dose of 7.2 mg/kg body weight twice a week by i.p. injection for 24 weeks, then followed by a feeding diet for 21 weeks. TNAP-treated mice showed an increase in mortality and time-dependent appearance of lesions in the liver. TNAP is hepatotoxic as demonstrated by a marked increase in the activities of serum alanine transaminase (ALT) and aspartic transaminase (AST). TNAP-related lesions observed histologically in mice, included hapatic atrophy, mild fatty metamorphosis with multilocular cysts in the liver. In conclusion, TNAP was considered to be a toxic compound in mice as evidenced by increased incidences of mortality, and lesions of liver.

Hibiscus protocatechuic acid protects against oxidative damage induced by tert-butylhydroperoxide in rat primary hepatocytes.

Hibiscus protocatechuic acid (PCA), a simple phenolic compound isolated from Hibiscus sabdariffa L., was studied for its protective effects against oxidative damage induced by tert-butylhydroperoxide (t-BHP) in a primary culture of rat hepatocytes. It had been reported that exposure of isolated hepatocytes to t-BHP results in leakage of lactate dehydrogenase (LDH) and alanine transaminase (ALT), peroxidation of cellular lipids, and depolarization of mitochondria. The present investigations showed that PCA at concentrations of 0.05 mg/ml and 0.10 mg/ml significantly decreased the leakage of LDH (P < 0.01) and ALT (P < 0.05 and P < 0.01) and the formation of malondialdehyde (MDA; P < 0.05 and P < 0.01) induced by 30-min treatment with t-BHP (1.5 mM) in primary cultured rat hepatocytes. PCA also attenuated t-BHP (0.10 mM) induced mitochondrial depolarization as determined by a retention test of rhodamine 123 and DNA repair synthesis as evidenced by unscheduled DNA synthesis (UDS). In addition, PCA exhibited an effective ability to quench 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH). In conclusion, PCA demonstrated protective effects against cytotoxicity and genotoxicity of hepatocytes induced by t-BHP. One of mechanisms of PCA's protective effect may be associated with its property of scavenging free radicals.