The toxicity and pharmacokinetics of dihydrosanguinarine in rat: a pilot study.

The quaternary benzo[c]phenanthridine alkaloid sanguinarine (SG) is the main component of Sangrovit, a natural livestock feed additive. Dihydrosanguinarine (DHSG) has recently been identified as a SG metabolite in rat. The conversion of SG to DHSG is a likely elimination pathway of SG in mammals. This study was conducted to evaluate the toxicity of DHSG in male Wistar rats at concentrations of 100 and 500 ppm DHSG in feed for 90 days (average doses of 14 and 58 mg DHSG/kg body weight/day). No significant alterations in body or organ weights, macroscopic details of organs, histopathology of liver, ileum, kidneys, tongue, heart or gingiva, clinical chemistry or hematology markers in blood in the DHSG-treated animals were found compared to controls. No lymphocyte DNA damage by Comet assay, formation of DNA adducts in liver by 32P-postlabeling, modulation of cytochrome P450 1A1/2 or changes in oxidative stress parameters were found. Thus, repeated dosing of DHSG for 90 days at up to 500 ppm in the diet (i.e. approximately 58 mg/kg/day) showed no evidence of toxicity in contrast to results published in the literature. In parallel, DHSG pharmacokinetics was studied in rat after oral doses 9.1 or 91 mg/kg body weight. The results showed that DHSG undergoes enterohepatic cycling with maximum concentration in plasma at the first or second hour following application. DHSG is cleared from the body relatively quickly (its plasma levels drop to zero after 12 or 18 h, respectively).

Natural feed additive of Macleaya cordata: safety assessment in rats a 90-day feeding experiment.

Macleaya cordata (Willd.) (Papaveraceae) is used as an active component in the natural feed additive Sangrovit. Sangrovit contains mixture of the intact aerial parts and the fraction of quaternary benzo[c]phenanthridine alkaloids from M. cordata (FQBA). In a 90-day pilot toxicity trial, Sangrovit and the FQBA were tested for safety. Male Wistar rats were fed for 90 days with 100, 7000 or 14000mg of Sangrovit or 600mg of FQBA in 1kg of feed. Body and organ weights, clinical chemistry and hematology markers, oxidative stress parameters, morphological structure of tongue, liver, ileum, kidney and heart samples, and total cytochrome P450 in liver were monitored. The results showed no statistically significant alterations in any parameter between control and treated animals, except for the group treated with 14000ppm Sangrovit that resulted in elevation of reduced glutathione level and superoxide dismutase activity in liver.

Flavonolignans from Silybum marianum moderate UVA-induced oxidative damage to HaCaT keratinocytes.

BACKGROUND: UV radiation from sunlight is a very potent environmental risk factor in the pathogenesis of skin cancer. Exposure to UV light, especially the UVA part, provokes the generation of reactive oxygen species (ROS), which induce oxidative stress in exposed cells. Topical application of antioxidants is a successful strategy for protecting the skin against UV-caused oxidative damage. OBJECTIVE: In this study, silybin (SB) and 2,3-dehydrosilybin (DS) (1-50 micromol/l), flavonolignan components of Silybum marianum, were tested for their ability to moderate UVA-induced damage. METHODS: Human keratinocytes HaCaT were used as an appropriate experimental in vitro model, to monitor the effects of SB and DS on cell viability, proliferation, intracellular ATP and GSH level, ROS generation, membrane lipid peroxidation, caspase-3 activation and DNA damage. RESULTS: Application of the flavonolignans (1-50 micromol/l) led to an increase in cell viability of irradiated (20 J/cm(2)) HaCaT keratinocytes. SB and DS also suppressed intracellular ATP and GSH depletion, ROS production and peroxidation of membrane lipids. UVA-induced caspases-3 activity/activation was suppressed by treatment with SB and DS. Lower concentrations of both compounds (10 micromol/l) significantly reduced cellular DNA single strand break formation. CONCLUSION: Taken together, the results suggest that these flavonolignans suppress UVA-caused oxidative stress and may be useful in the treatment of UVA-induced skin damage.

Attenuation of UVA-induced damage to human keratinocytes by silymarin.

BACKGROUND: UV radiation from sunlight is a potent environmental risk factor in skin cancer pathogenesis. UVA is the major portion of UV light reaching the earth surface ( approximately 95%) and it is reported to lead to benign and malignant tumor formation. UVA-mediated cellular damage occurs primarily through the release of reactive oxygen species (ROS) and it is responsible for inflammation, immunosuppression, photoaging and photocarcinogenesis. OBJECTIVE: The aim of our study was to investigate the potency of silymarin, the polyphenol fraction from the seeds of Silybum marianum, to modulate UVA-induced oxidative damage to human keratinocytes. METHODS: Skin epidermal cell line HaCaT, extensively used for studying the influence of UV radiation, was chosen as an experimental model. Silymarin's effect on UVA-disrupted cell viability, proliferation, mitochondrial function, and intracellular ATP and GSH level was measured. Furthermore, silymarin's potency to reduce UVA-induced ROS generation, membrane lipid peroxidation, caspase-3 activation and DNA damage was monitored. RESULTS: Treatment of irradiated HaCaT (20 J/cm(2)) with silymarin (0.7-34 mg/l; 4h) resulted in concentration-dependent diminution of UVA-caused oxidative stress on all studied parameters. Silymarin application extensively reduced GSH depletion and ROS production as well as lipid peroxidation in irradiated cells. Formation of UVA-induced DNA single strand breaks and caspase-3 activity was also significantly decreased by silymarin. CONCLUSION: The results suggest that silymarin may be beneficial in the treatment of UVA-induced skin oxidative injury and inflammation. However, further studies especially whose using human systems are needed to determine efficacy of silymarin in vivo.

Cytotoxicity of sanguinarine in primary rat hepatocytes is attenuated by dioxin and phenobarbital.

Putative interactions between quaternary benzo[c]phenanthridine alkaloid sanguinarine (SA) and aryl hydrocarbon receptor/cytochrome P450 CYP1A (AhR/CYP1A) regulatory pathway are the subject of perpetual disputations. The role of CYP1A enzymes and AhR receptor in SA cytotoxicity was anticipated. In this paper, we tested, whether selected inducers of CYP enzymes modulate cytotoxicity of SA in primary cultures of rat hepatocytes. Cells were challenged 48h with dioxin (TCDD; 5nM), phenobarbital (PB; 500microM) or DMSO prior to the treatment with SA. SA itself displayed time- and dose-dependent cytotoxicity as revealed by lactate dehydrogenase leakage into the medium and MTT test. Pre-treatment of hepatocytes with TCDD and/or PB significantly attenuated SA cytotoxicity, the effects being more pronounced at lower concentrations of SA and shorter periods of incubation. We assumed involvement of CYP1A enzymes in diminution of SA cytotoxicity. Surprisingly, co-treatment with SA and furafylline, an inhibitor of CYP1A enzymes, further attenuated SA cytotoxicity instead of expected reversal of this effect. We conclude that TCDD- and PB-inducible genes attenuate cytotoxicity of SA in rat hepatocytes. CYP1A enzymes are not involved in this attenuation, but they rather augment SA cytotoxicity. Future research should focus on analyses of the involvement of other CYPs in SA cytotoxicity and on identification of TCDD-/PB-controlled genes responsible for observed phenomenon.

Effects of sanguinarine and chelerythrine on the cell cycle and apoptosis.

OBJECTIVES: This review summarizes the involvement of sanguinarine and chelerythrine in cell cycle regulation and cell death in various cell lines. It is focused on their potential in the treatment of cancer. METHODS: We conducted a search of PubMed, ScienceDirect and Medline for papers on the molecular mechanisms of the biological activity of sanguinarine and chelerythrine published mainly from 1995 to 2006. RESULTS AND CONCLUSIONS: Our analysis of the published studies suggested that these alkaloids are not only good candidates for chemotherapeutic regimens but may also contribute to the development of successful immune therapies of some carcinomas due to their apoptotic potential. However, the complete signalling cascade in which sanguinarine and chelerythrine treatment induces apoptotic cell death is not yet understood. Overall, the results of recent studies suggest that sanguinarine and chelerythrine may be useful as agents in the management of cancer.

Activation of the aryl hydrocarbon receptor by berberine in HepG2 and H4IIE cells: Biphasic effect on CYP1A1.

Berberine has long been considered a candidate for an antimalarial drug. It exerts a plethora of biological activities and has been used in the treatment of diarrhea and gastro-enteritis for centuries. Here we provide evidence that berberine activates the aryl hydrocarbon receptor (AhR) in human hepatoma (HepG2) and rat hepatoma cells stably transfected with a dioxin responsive element fused to the luciferase gene (H4IIE.luc). AhR was activated by high doses of berberine (10-50 microM) after 6 and 24 h of incubation as revealed by CYP1A1 mRNA expression (HepG2) and AhR-dependent luciferase activity (H4IIE.luc). Berberine induced nuclear translocation of AhR-GFP chimera transiently transfected to Hepa1c1c7 cells. In contrast, low doses of berberine (<1 microM) and prolonged times of the treatments (48 h) failed to produce any activation of AhR in H4IIE.luc cell line. HPLC analysis ruled out the hypothesis that the loss of berberine capacity to activate AhR in H4IIE.luc cells is due to metabolic inactivation of the alkaloid. We demonstrate that berberine is a potent inhibitor (IC50=2.5 microM) of CYP1A1 catalytic activity (EROD) in HepG2 cell culture and in recombinant CYP1A1 protein. Collectively, our results imply that while berberine activates the Ah receptor, it is accompanied by inactivation of the catalytic activity of CYP1A1 and occurs at concentrations that exceed those predicted to occur in vivo. Given these data, it appears that activation of the AhR pathway by berberine has a low toxicological potential.

Prunella vulgaris extract and rosmarinic acid prevent UVB-induced DNA damage and oxidative stress in HaCaT keratinocytes.

Solar radiation is a very important exogenous factor in skin pathogenesis and can lead to the development of a number of skin disorders. UVB irradiation is known to induce oxidative stress, inflammation and especially DNA lesions in exposed cells. It is important, therefore, to identify agents that can offer protection against UVB-caused skin damage. Natural compounds have been studied for their possible ability to control/modulate various lifestyle-related diseases. The application of plant compounds/extracts with screening, antioxidant and anti-inflammatory activities may also successfully protect the skin against UV-caused injury. We assessed the potency of Prunella vulgaris extract (PVE) and its main phenolic acid component, rosmarinic acid (RA), to suppress UVB-induced (295-315 nm) alterations to human keratinocytes HaCaT using a solar simulator. Pre- and post-treatment of HaCaT cells with PVE (5-50 mg/l) and RA (0.18-1.8 mg/l) reduced breakage together with the apoptotic process. PVE and RA also significantly eliminated ROS production and diminished IL-6 release. Taken together, both PVE and RA prevent UVB-caused injury to keratinocytes. However their efficacy needs to be demonstrated in vivo.

Effect of different calcineurin inhibitors on AOPP and TAS after kidney transplantation.

OBJECTIVES: Little is known about the influence of calcineurin inhibitors on advanced oxidation protein products (AOPP) and total antioxidant status (TAS) after renal transplantation. DESIGN AND METHODS: AOPP and TAS were evaluated in transplanted patients on different calcineurin inhibitors. Thirty-five patients were treated with cyclosporine A (group A) and 33 with tacrolimus (group B). RESULTS: Over 6 months, the mean levels of AOPP in group A decreased from 205.9+/-125.7 to 140.9+/-78.9 micromol/L and TAS from 1.89+/-0.30 to 1.75+/-0.27 mmol/L. In group B, the mean levels of AOPP decreased from 196.5+/-123.9 to 129.6+/-63.8 micromol/L and TAS from 1.80+/-0.39 to 1.78+/-0.23 mmol/L. CONCLUSION: No significant differences in AOPP and TAS were found with respect to treatment. The only exception was the higher mean concentration of AOPP at month 1 in group A (p=0.026).

Long-term effects of three commercial cranberry products on the antioxidative status in rats: a pilot study.

Cranberry (Vaccinium macrocarpon Ait. Ericaceae) fruits and juice are widely used for their antiadherence and antioxidative properties. Little is known however about their effects on clinical chemistry markers after long-term consumption. This study was conducted to evaluate the effect of three commercial cranberry products, NUTRICRAN90S, HI-PAC 4.0, and PACRAN on the antioxidative status of rodents, divided into three experimental groups. The products were given as dietary admixtures (1500 mg of product/kg of stock feed) for 14 weeks to male Wistar rats (Groups 2-4) and a control Group 1 which received only stock feed. There were no significant cranberry treatment-related effects on oxidative stress parameters, catalase, glutathione peroxidase, glutathione reductase, glutathione transferase, superoxide dismutase, total antioxidant capacity, thiobarbituric acid reactive substances, advanced oxidation protein products, total SH-groups, or any other measured clinical chemistry markers. Hematological parameters, body weight, and food consumption were also unaffected by intake of cranberries. Only liver glutathione reductase activity and glutathione levels were significantly lower in Group 4 than in Group 1. Plasma alkaline phosphatase alone was significantly decreased in Group 2. No gross pathology, effects on organ weights, or histopathology were observed. No genotoxicity was found, and total cytochrome P450 level in liver was unaffected in all groups. The levels of hippuric acid and several phenolic acids were significantly increased in plasma and urine in Groups 2-4. The concentration of anthocyanins was under the detection threshold. The dietary addition of cranberry powders for 14 weeks was well tolerated, but it did not improve the antioxidative status in rats.

Lonicera caerulea and Vaccinium myrtillus fruit polyphenols protect HaCaT keratinocytes against UVB-induced phototoxic stress and DNA damage.

BACKGROUND: Sunlight is a very potent environmental factor in skin pathogenesis and can induce skin cancer. UVB irradiation is known to cause oxidative stress, inflammation and especially DNA damage. Topical application of agents with UV absorbing, antioxidant and anti-inflammatory activities is a successful strategy in the protection of the skin against UV-caused damage. OBJECTIVE: To examine the ability of the phenolic fraction of Lonicera caerulea and Vaccinum myrtillus fruits to moderate UVB-induced damage. METHODS: HaCaT keratinocytes, a well-established in vitro system for investigations on UV radiation induced cell damage, were used to assess the effects of pre- and post-treatment with L. caerulea (LCE) and V. myrtillus (VME) phenolic fractions (5-50 mg/l) on keratinocyte damage induced by a solar simulator (295-315 nm). RESULTS: In this study, a model of UVB-induced damage to HaCaT was established. LCE and VME efficiently reduced the extent of DNA breakage (especially at concentrations of 25 and 10 mg/l) together with caspase-3 and -9 activity and DNA laddering induced by UVB (100 or 200 mJ/cm(2)). LCE and VME significantly decreased RONS generation and partially diminished IL-6 expression. LCE pre-treatment also prevented keratinocytes proliferation. CONCLUSION: The results suggest that the phenolic fraction of L. caerulea and V. myrtillus fruits suppress UVB-caused injury to keratinocytes. These results now need to be demonstrated in vivo.

Macleaya cordata extract and Sangrovit genotoxicity. Assessment in vivo.

BACKGROUND: Sanguinarine (SG) has been reported to form DNA adducts in vitro and increase the levels of DNA single strand breaks in the blood and bone marrow of mice treated intraperitoneally with SG. Recently, we showed no genotoxic effects of orally administrated 120 mg/kg feed Macleaya cordata extract (a mixture of sanguinarine and chelerythrine) in pigs or rats in 90-day studies. The goal of this paper was to assess the possible genotoxicity of M. cordata extract when included as a dietary admixture to rodents at concentrations providing 600 mg/kg feed and 100, 7000 or 14000 mg/kg feed Sangrovit (natural feed additive containing M. cordata extract and powdered M. cordata) in a 90-day pilot study. METHODS AND RESULTS: The rats consumed ad libitum either the standard diet or the diets containing 367 ppm of sanguinarine and chelerythrine in M. cordata extract, and 5, 330, or 660 ppm of total alkaloids in Sangrovit for 90 days. The DNA adducts formation in liver was analyzed by (32)P-postlabeling technique and DNA single strand breaks in lymphocytes were evaluated by Comet assay. The results showed that M. cordata extract and/or Sangrovit induced no DNA damage to rat lymphocytes or hepatocytes after 90-days oral administration. CONCLUSIONS: Data from the studies described in this paper and the fact that Sangrovit given to the rats in our experiments were higher than the recommended dose (50 to 100 mg/kg feed), argue strongly in favour of the use of Sangrovit in live stock.