Treatment of benign prostatic hyperplasia with Croton membranaceus in an experimental animal model.

ETHNOPHARMACOLOGICAL RELEVANCE: Croton membranaceus leaf extracts are used in the Bahamas to aromatize tobacco. In Nigeria it is used to improve digestion and in Ghana, the root extract is used for the treatment of benign prostatic hyperplasia (BPH). Despite claims of efficacy no data exists to support this. The aim of this study was to determine if Croton membranaceus aqueous root extract (CMARE) could attenuate the development of BPH in an animal model. MATERIALS AND METHODS: Fifty (50) adult male Sprague-Dawley rats weighing 200-250g were randomly divided into 5 groups. Group 1 served as the control and received normal saline p.o. Groups 2-5 were castrated and injected with 5mg/kg b.wt. testosterone propionate subcutaneously for 28 days. Group 2 (model group) had no further treatment. Group 3 was simultaneously given 0.5mg/kg b.wt. finasteride p.o. throughout. Groups 4 and 5 received 30mg/kg b.wt. [low dose (LD)] and 300mg/kg b.wt. [high dose (HD)] CMARE, respectively, for 28 days. Rats were sacrificed at the end of the study and all prostate organs harvested. Wet weights, volumes and prostatic index (PI) were determined. Tissues were histologically examined. Serum prostate specific antigen (PSA) and dihydrotestosterone (DHT) levels were determined. RESULTS: Prostate volume of the control group was 0.67±0.23cm(3). The model, finasteride, CMARE LD and HD groups had the following volumes: 0.92±0.12, 0.84±0.16, 0.79±0.16 and 0.80±0.19cm(3), respectively. Only the model group showed significant statistical differences with the control (p=0.007). PI for control, model, finasteride, LD and HD groups was as follows: 0.19±0.04, 0.30±0.04, 0.25±0.04, 0.21±0.05 and 0.22±0.05. No statistical differences between the control PI and the CMARE treated groups were observed. Histologically, the model group had massive growth of columnar stromal and epithelial cells. CMARE and finasteride attenuated this growth with a resultant thin layer of stromal and epithelial cells similar to the control. PSA levels were significantly lower in the treatment groups. CONCLUSION: CMARE reduces stromal and epithelial cell growth, and subsequently shrinks enlarged prostate. This is the first scientific proof validating the anecdotal evidence of CMARE efficacy in the management of BPH.

Mutagenicity of edible palm oil on the Ghanaian market before and after repeated heating.

Red palm oil produced in Ghana largely by village folks has never been tested for its mutagenic potential. The study aimed at determining the mutagenicity of high-energy heated red palm oil (RRPO) and refined, bleached imported palm oil (PO) on the Ghanaian market. Samples of RRPO and PO were 1× and 5× heated for 10 min at 180 °C with a cooling period of 5 h in-between. Unheated, together with heated samples, were tested for mutagenicity using Salmonella typhimurium TA 98 and TA 100 tester stains. Unheated PO was negative for the Ames mutagenicity test with TA 98 strain. However, 1× and 5× heated PO were mutagenic (P = 0.05, each). Testing PO, using TA 100 strain was negative. RRPO was mutagenic with TA 98 strain for heated oils (P = 0.05, each). Assays with TA 100 strain showed highly significant mutations (P = 0.001, each) that increased with increasing heating frequency. PO 1× and 5× heated samples caused significant frameshift mutation in the S. typhimurium TA 98 strain. RRPO caused highly significant point and frameshift mutations in heated samples. Furthermore, unheated RRPO mutagenic potential has serious health implications.

Acute toxicity studies of Croton membranaceus root extract.

AIM OF THE STUDY: Croton membranaceus root and leaf extracts are used in the Bahamas to aromatize tobacco, in Nigeria to improve digestion, and in Ghana, for benign prostate hyperplasia. Despite claims of success there is paucity of information on its toxicity. The aim of this study was to determine if Croton membranaceus has acute toxicity properties. MATERIALS AND METHODS: Roots were air-dried in a solar dryer for one week before milling. The powder was extracted with 96% ethanol, freeze-dried and re-extracted with distilled water and freeze-dried. 15 male Sprague-Dawley rats (180-200 g) were divided equally into 2 treatment groups [low dose (LD) and high dose (HD)], plus a control group (C). LD and HD received 1500 and 3000 mg/kg b.wt. Croton membranaceus aqueous extract, respectively, one time and observed for 14 days. Haematological [Full Blood Count and haemoglobin (Hb)], biochemical [bilirubin, alanine aminotransferase (ALA), aspartate aminotransferase (AST), total protein, albumin, globulin, alkaline phosphatise (ALP), γ-glutamyltranspetidase (GGT), urea, creatinine, creatinine kinase - Muscle and Brain (CK-MB), creatinine kinase - Total (CK-R)] examinations were performed. RESULTS: Control group's CK-MB (5444±534 U/L) and LD group CK-MB (4014±1016 U/L) were significantly different (p<0.05). Control and the HD group CK-MB (3955±1135 U/L) were significantly different (p<0.05). Both LD and HD CK-R levels (697±197U/L and 732±203 U/L, respectively), were lower than the control (1139±220 U/L) at 48 h and 14 days (p<0.05, p<0.05, respectively). γ-GT levels of the HD group was 4.8±0.4 U/L compared to the Control group value of 0.9±0.2 U/L (p<0.05). CONCLUSIONS: Taking all factors into consideration, Croton membranaceus ingestion does not produce general acute toxicity. However, its creatinine kinase lowering ability could be explored.

Possible adverse effect of high delta-alpha-tocopherol intake on hepatic iron overload: enhanced production of vitamin C and the genotoxin, 8-hydroxy-2'- deoxyguanosine.

Excess hepatic iron generates reactive oxygen species that result in oxidative stress and oxidative damage to the liver. Vitamins have hitherto been considered to be a possible remedy. The aim of this study was to determine if high doses of delta-alpha-tocopherol supplementation in iron overload would ameliorate the oxidative stress. Four groups of 20 male Wistar albino rats were studied: group 1 (control) was fed normal diet, group 2 (Fe) 0.75% Ferrocene iron, group 3 (FV gp) 0.75% Ferrocene/delta-alpha-tocopherol (10x RDA), group 4 (V gp) normal diet/delta-alpha-tocopherol. After 12 months, serum iron, reduced glutathione, catalase, vitamin C, Oxygen Radical Absorbance Capacity, lipid peroxidation, 8-hydroxy-2'-deoxyguanosine (8-OHdG), aspartate transaminase (AST), and alanine transaminase (ALT) were measured. Vitamin C levels were: F gp = 5.04 +/- 0.09; FV gp = 5.85 +/- 0.13 (micromol/l) (p < 0.05). 8-hydroxy-2'-deoxyguanosine levels were: F gp = 143.6 +/- 6.4; FV gp = 179.2 +/- 18.2 (ng/ml) (p < 0.05). Oxidative liver damage, as determined by serum AST and ALT levels, was not attenuated by alpha-tocopherol. A positive correlation existed between vitamin C and 8-OHdG, suggesting possible delta-alpha-tocopherol toxicity.

Serum levels of Cu, Se, and Zn in adult rural/urban residents in Ghana: paradigm shift?

Deficiencies in Cu, Se, and Zn impair one or more biochemical functions, and excess are associated with toxicity. Baseline studies on the Ghanaian population are scanty. The study was undertaken to determine whether significant rural/urban differences in the serum levels of Cu, Se, and Zn did exist. Forty males/60 females from rural and 50 males/50 females from urban Ghanaian communities were sampled. Serum Cu, Se, and Zn were determined using flame atomic absorption spectrometry. Cu level for rural and urban subjects was 997 +/- 333 and 979 +/- 290 microg/L, respectively (p = 0.68). However, Cu levels were significantly higher in the rural females (1,063 +/- 367 microg/L) than the rural males (898 +/- 249 microg/L; p = 0.0085). Se levels for rural/urban subjects were 97 +/- 36 and 87 +/- 31 microg/L, respectively (p = 0.03). Zn levels in the rural/urban subjects were 312 +/- 218 and 150 +/- 102 microg/L, respectively (p = 0.002). Additionally, Zn was significantly higher in rural females (428 +/- 204 microg/L) than the urban females (166 +/- 103 microg/L; p = 0.0002). Finally, Zn was significantly higher in rural females (428 +/- 204 microg/L) than males (172 +/- 116 microg/L; p = 0.0028). In conclusion, Cu, Se, and Zn were higher in the rural group compared to the urban group, and the generally low Zn levels were confirmed in another cohort follow-up study.

Synergistic interaction between excess hepatic iron and alcohol ingestion in hepatic mutagenesis.

BACKGROUND/AIM: Hereditary hemochromatosis (HH) and dietary iron overload are the main iron-loading diseases. Fibrosis, cirrhosis and hepatocellular carcinoma (HCC) are complications to HH and dietary iron overload possibly influenced by co-factors. Alcohol may be one such factor. The aim therefore was to determine the extent of synergistic interaction between free hepatic iron and alcohol, complicating dietary iron overload in HCC pathogenesis. METHODS: Four groups of 20 Wistar albino rats were used: group 1 (C) was fed the chow diet; group 2 (Fe) was supplemented with 0.75% ferrocene iron; group 3 (Fe+Al), 0.75% iron and 7% ethanol; and group 4, 7% ethanol (Al) for 12 months. Iron profile, superoxide/nitrite free radicals, lipid peroxidation (LPO)/8-isoprostane (8-IP), 8-hydroxydeoxyguanosine (8-OHdG), oxidative lipid/DNA damage immunohistochemistry, transaminases (AST/ALT) and Ames mutagenesis tests were performed. RESULTS: Significant differences were observed in the Fe+Al group for LPO, 8-IP, AST and ALT (p<0.001, 0.001, 0.001 and 0.001, respectively) compared to other groups. A three-fold synergistic interaction was observed for the same parameters. Furthermore, significant differences of p<0.05 and 0.001 were observed for 8-OHdG and mutagenesis, respectively, with an additive synergy in the Fe+Al group. ALT/8-OHdG and ALT/mutagenesis correlated positively (p<0.04 and 0.008, respectively). The immunohistochemistry revealed iron/alcohol multiplicative synergism with hydroxyl radical involvement. CONCLUSION: Mutagenic effects of iron and alcohol are synergistically multiplicative implicating hydroxyl free radicals in hepatocarcingenesis.

Interactions between aflatoxin B1 and dietary iron overload in hepatic mutagenesis.

BACKGROUND/AIM: Dietary aflatoxin B(1) (AFB(1)) exposure and iron overload are important causes of hepatocellular carcinoma in sub-Saharan Africa. The aim of this study was to investigate if the two risk factors have an interactive effect. METHODS: Four groups of Wistar albino rats were studied for 12 months. Group 1 (control) was fed the normal chow diet; group 2 (Fe) was supplemented with 0.75% ferrocene iron; group 3 (Fe+AFB(1)) was fed 0.75% ferrocene throughout and gavaged 25 microg AFB(1) for 10 days; group 4 (AFB(1)) was gavaged 25 microg AFB(1) for 10 days. Iron profile, lipid peroxidation (LPO), 8-hydroxydeoxyguanosine (8OHdG), oxidative lipid/DNA damage immunohistochemistry, superoxide/nitrite free radicals, cytokines IL6, IL-10, transaminases (ALT/AST) and Ames mutagenesis tests were performed. RESULTS: LPO and ALT showed a significant (p<0.05)/additive effect and 8OHdG a significant (p<0.05)/multiplicative effect in the Fe+AFB(1) group. IL-6 produced a negative synergy as against an additive antagonistic effect with IL-10. Massive deposits of 4-hydroxynonenal (4-HNE) and 8OHdG were observed in liver sections of the Fe+AFB(1) group, suggestive of multiplicative synergy. Significant levels of mutagenesis (p<0.001) were observed in the Fe+AFB(1) group. This multiplicative synergy was five-fold. CONCLUSION: Dietary iron overload and AFB(1) have a multiplicative effect on mutagenesis.

Hepatocellular carcinoma caused by iron overload: a possible mechanism of direct hepatocarcinogenicity.

BACKGROUND/AIMS: Excess hepatic iron may be both directly and indirectly carcinogenic. The aim of this study was to determine if generation of reactive oxygen species and the resulting oxidative damage induced by free hepatic iron is directly hepatocarcinogenic. METHODS: Sixty male Wistar albino rats were iron-loaded by ferrocene supplementation of their diet. Biochemical parameters of oxidative damage and lipid peroxidation, DNA unwinding and strand breaks, and the Ames Mutagenesis Test were measured at 4 monthly intervals and correlated with the degree of hepatic iron overload, the presence of iron-free preneoplastic foci in the liver, and the development of hepatocellular carcinoma in comparison with 60 control rats. RESULTS: Levels of lipid hydroperoxides, malonaldehyde, 8-isoprostane and 8-hydroxy-2'-deoxyguanosine increased, reaching peak concentrations at 20-24 months, and correlating with an increase in the rate of DNA unwinding, strand breaks, and positive Ames Tests. Iron-free neoplastic foci became evident at 16 months and thereafter increased in number. Preneoplastic foci were present in five of eight rats remaining at 32 months and HCC had developed in one of the five. CONCLUSIONS: Our findings are compatible with the hypothesis that the direct hepatocarcinogenic effect of free iron is mediated by the generation of oxygen reactive species and oxidative damage that are mutagenic and carcinogenic.

Iron-free neoplastic nodules and hepatocellular carcinoma without cirrhosis in Wistar rats fed a diet high in iron.

Although excess hepatic iron in hereditary haemochromatosis and dietary iron overload in the African causes hepatocellular carcinoma, it usually does so in the presence of cirrhosis. A direct hepatocarcinogenic effect of iron has not been proved. Moreover, an animal model of hepatocellular carcinoma induced by iron overload has not been available. The aim of this study was to develop such a model and to use it to ascertain whether excess hepatic iron is directly hepatocarcinogenic. Sixty Wistar albino rats were fed a chow diet and 60 the same diet supplemented initially with 2% carbonyl iron for 12 months, followed by 0.5% ferrocene for 20 months. Five rats from each group were sacrificed every 4 months for 24 months for histological and biochemical monitoring. By 16 months, hepatocytes in all the rats receiving the iron-supplemented diet showed grade 4 iron overload, comparable in degree with that seen in patients with advanced hereditary haemochromatosis and dietary iron overload. Altered hepatic foci and pre-neoplastic nodules were first seen at 16 months. These increased in size and number with time, were iron-free, stained positively with placental glutathione sulphydryl transferase, and showed the same histological features as the iron-free foci described in patients with hepatocellular carcinoma complicating hereditary haemochromatosis. At 32 months the eight surviving rats in the iron overloaded group were sacrificed. The livers of five of these rats contained pre-neoplastic nodules and one showed, in addition, an iron-free, well-differentiated hepatocellular carcinoma. The tumour stained positively for placental glutathione sulphydryl transferase. Neither cirrhosis nor portal fibrosis was present in this or any iron-loaded animal. We conclude that hepatocellular carcinoma may complicate dietary hepatic iron overload in Wistar albino rats in the absence of fibrosis or cirrhosis, confirming an aetiological association between dietary iron overload and the tumour and suggesting that iron may be directly hepatocarcinogenic.