Altered lipid profile, oxidative status and hepatitis B virus interactions in human hepatocellular carcinoma.

Altered membrane integrity in hepatocellular carcinoma (HCC) tissue was indicated by an elevation in cholesterol and significant decrease in phosphatidylcholine (PC). The resultant decreased phosphatidylcholine/phosphatidylethanolamine (PC/PE) and increased cholesterol/phospholipid ratios are associated with decreased fluidity in the carcinoma tissue. The lower PC was associated with a decrease in the quantitative levels of the saturated (C16:0, C18:0), omega6 (C18:2, C20:4) and omega3 (C22:5, C22:6) fatty acids (FAs), resulting in reduced long-chain polyunsaturated fatty acids (LCPUFAs), total PUFA and an increase in omega6/omega3 FA ratio. In PE, the saturated and omega3 (C22:5, C22:6) FAs were reduced while the total omega6 FA level was not affected, leading to an increased omega6/omega3 FA ratio. Increased levels of C18:1omega9, C20:2omega6 and reduction of 22:6omega3 in PC and PE suggest a dysfunctional delta-6 desaturase. The reduced PC/PE ratio resulted in a decreased C20:4omega6 (PC/PE) ratio, implying a shift towards synthesis of the 2-series eicosanoids. Lipid peroxidation was reduced in both hepatitis B negative (HBV(-)) and positive (HBV(+)) HCC tissues. Glutathione (GSH) was decreased in HCC while HBV had no effect, suggesting an impairment of the GSH redox cycle. In contrast HBV infection enhanced GSH in the surrounding tissue possibly to counter oxidative stress as indicated by the increased level of conjugated dienes. Apart from the reduced LCPUFA, the low level of lipid peroxidation in the carcinoma tissue was associated with increased superoxide dismutase and glutathione peroxidase activity. The disruption of the redox balance, resulting in increased cellular antioxidant capacity, could create an environment for resistance to oxidative stress in the carcinoma tissue. Alterations in membrane cholesterol, phospholipids, FA parameters, C20:4omega6 membrane distribution and low lipid peroxidation are likely to be important determinants underlying the selective growth advantage of HCC cells.

Exposure assessment for fumonisins in the former Transkei region of South Africa.

The fumonisins are mycotoxins produced mainly by Fusarium verticillioides and F. proliferatum in maize, the predominant cereal staple for subsistence farming communities in southern Africa. In order to assess exposure to these mycotoxins in the Bizana (now known as Mbizana) and Centane magisterial areas of the former Transkei region of the Eastern Cape Province of South Africa, the actual maize consumption by different age groups in these communities was measured. In the groups 1-9 years (n = 215) and 10-17 (n = 240) years, mean consumption (+/-standard error) was 246 +/- 10.8 and 368 +/- 10.3 g per person day(-1), respectively, with no significant difference (p > 0.05) between the magisterial areas. For adults (18-65 years) mean maize consumption in Bizana (n = 229) and Centane (n = 178) were significantly different (p < 0.05) at 379 +/- 10.5 and 456 +/- 11.9 g per person day(-1), respectively. An exposure assessment was performed by combining the maize consumption distribution with previously determined levels of total fumonisin (fumonisins B(1) and B(2) combined) contamination in home-grown maize in these two areas. Assuming an individual adult body weight of 60 kg, fumonisin exposure in Bizana, an area of relatively low oesophageal cancer incidence, was 3.43 +/- 0.15 microg kg(-1) body weight day(-1), which was significantly lower (p < 0.05) than that in Centane (8.67 +/- 0.18 microg kg(-1) body weight day(-1)), an area of high oesophageal cancer incidence. Mean fumonisin exposures in all age groups in both Bizana and Centane were above the provisional maximum tolerable daily intake (PMTDI) of 2 microg kg(-1) body weight day(-1) set by the Joint FAO/WHO Expert Committee on Food Additives.

Toxicity of culture material of Fusarium verticillioides strain MRC 826 to nonhuman primates.

We conducted a chronic feeding study in vervet monkeys (Cercopithecus aethiops) over 13.5 years. The experimental design consisted of two dietary treatment groups, each including males and females, fed varying levels of culture material of Fusarium verticillioides (Sacc.) Nirenberg (= F. moniliforme Sheldon) strain MRC 826 mixed into their daily food ration. Two females were included as treatment controls. We conducted blood chemical analyses bimonthly and recorded all clinical signs during the course of the experiment. We took liver biopsies at various stages during the initial phase of the experiment. Several monkeys were terminated in extremis during the experiment. Detailed feed intake profiles were determined 5 years after the experiment began, and the fumonisin B (FB) mycotoxin content of the feed was determined during the final stages of the experiment. The apparent FB consumption patterns were related to changes observed in the biochemical parameters in the blood and urine, including the liver function enzymes and creatinine clearance as well as differential blood counts and sphingolipid levels in the serum and urine. An apparent no-effect threshold for kidney and liver damage is estimated to be between 0.11 and 0.18 mg FB/kg body weight (bw)/day, which corresponds to a feed contamination level of between 8.21 and 13.25 mg FB/kg bw diet. Apart from the effects on the liver and kidney, a wide variety of parameters, including cholesterol and creatine kinase, were also adversely affected. Several blood parameters, including white and red blood cells, also significantly decreased in the treated animals. The serum sphinganine level and the sphingosine/sphinganine ratio, monitored toward the end of the experiment, significantly increased in both the low-dose and high-dose animals. The present study provides important information about the diversity of lesions induced by culture material of F. verticillioides in vervet monkeys and the dosage levels of fumonisins to be used in long-term studies in nonhuman primates.

The effect of a single gavage dose of fumonisin B(2) on the sphinganine and sphingosine concentrations in vervet monkeys.

The disruption in sphinganine (Sa) and sphingosine (So) concentrations in plasma and urine of vervet monkeys (Cercopithecus aethiops) was measured following a single gavage dose of either 1 (low dose) or 10 mg (high dose) fumonisin B(2) (FB(2))/kg body weight. Blood and urine were collected over a 51-day period. In the low-dose monkeys, none of the parameters measured increased significantly above the control values. In the high-dose monkeys the plasma Sa/So ratios were significantly increased (P< 0.05) above the corresponding control ratios after 3 days and continued to be significantly raised for another 27 days, whereafter the ratios declined to control values after 51 days. The plasma aspartate transaminase (AST) activities increased significantly above their control values from day 5 to day 23 and the gamma-glutamyl transferase (GGT) activities from day 7 until the end of the study period. The plasma cholesterol concentration and alanine transaminase (ALT) and lactate dehydrogenase (LDH) activities increased transiently, but not significantly, and returned to control values within the study period. The urinary Sa/So ratio, plasma creatinine and urea values in both groups of monkeys did not increase above the control values. This study demonstrated that a single large dose of FB(2) can cause transient disruption of sphingoid metabolism in vervet monkeys.

The effect of repeated gavage doses of fumonisin B1 on the sphinganine and sphingosine levels in vervet monkeys.

Fumonisins occur widely around the world in maize products intended for human and animal consumption posing a health hazard. Direct measurement of fumonisins in physiological fluids as a biomarker of exposure is not practicable, however fumonisins disrupt sphingoid metabolism by inhibition of sphinganine N-acyltransferase. In this study the disruption in sphinganine (Sa) and sphingosine (So) levels in plasma and urine was measured in vervet monkeys (Cercopithecus aethiops) dosed with repeated gavages of 1mg fumonisin B1 (FB1)/kg body weight three times/week continuously over a 51-day period. The plasma Sa/So ratio reached a maximum (mean of 4.3) after 30 days in the dosed monkeys with a three-fold increase above the ratio of the control monkeys and then declined slowly to double the value in controls after 51 days. The lack of a clear elevation in urinary Sa/So ratios after 51 days of multiple exposure in the dosed monkeys indicates that the plasma ratio is more sensitive than urinary changes in monkeys. This is confirmed by the plasma levels of liver function enzymes of which aspartate transaminase, glutamyl-transferase and lactate dehydrogenase were increased in the dosed monkeys, while the plasma indicators of renal function were not increased above the levels in the control monkeys. Thus repeated low doses of FB1 can cause sustained disruption of sphingoid metabolism.

The effect of a single gavage dose of fumonisin B(1) on the sphinganine and sphingosine levels in vervet monkeys.

This is the first report of sphinganine (Sa) and sphingosine (So) levels determined in serum and urine of vervet monkeys (Cercopithecus aethiops) dosed with pure fumonisin B(1) (FB(1)). Initially, experimental vervet monkeys were given a single gavage dose of either 1 or 10 mg FB(1) /kg body weight. Blood and urine were sampled daily and on day seven the monkeys were terminated and the kidneys and livers harvested. In a subsequent experiment, other vervet monkeys were similarly dosed and blood and urine samples were collected over a 50-day period. In the high-dose monkeys the serum Sa/So ratio, as well as levels of serum cholesterol and liver function enzymes, increased during the first week after dosing and remained elevated for several weeks thereafter. The urinary Sa/So ratio and the serum renal function indicators showed a more rapid response and a correspondingly more rapid return to pre-dosing levels. In the low-dose monkeys serum Sa and the Sa/So ratio were the only parameters to increase above the control levels. The Sa/So ratio in liver and kidney tissue showed an elevation over controls in a dose-dependent manner. The serum Sa/So ratio was exclusively elevated above the control levels in the low- and high-dose monkeys and seems more relevant as a marker for fumonisin exposure than any of the other indicators.

Disruption of sphingolipid metabolism in non-human primates consuming diets of fumonisin-containing Fusarium moniliforme culture material.

The fumonisin mycotoxins are produced by Fusarium moniliforme Sheldon, a contaminant of corn worldwide. The two most abundant analogues (fumonisins B1 and B2) are known to be potent inhibitors of sphingosine N-acyltransferase (ceramide synthase) and hence to disrupt de novo sphingolipid biosynthesis. The sphingoid bases, sphingosine and sphinganine (and hence their ratio), were measured at varying intervals over a period of 60 weeks in the serum of non-human primates (vervet monkeys; Cercopithecus aethiops) which were consuming diets containing 'low' and 'high' amounts of F. moniliforme culture material, such that their total daily fumonisin intake was approximately 0.3 and 0.8 mg/kg body weight/day, respectively. Although no significant differences were found in the serum levels of sphingosine compared to controls, serum sphinganine levels in the experimental groups (mean of 219 nM and 325 nM, respectively) were significantly (P = 0.02) elevated above the levels in controls (mean 46 nM). As a consequence, the ratio sphinganine:sphingosine was significantly (P = 0.003) elevated from a mean of 0.43 in the control group to 1.72 and 2.57 in the experimental groups, respectively. Similar changes in sphingolipid profiles were also measured in urine with an increase of the ratio from 0.87 in controls to 1.58 and 2.17 in the experimental groups, although the differences were not statistically significant. Hence, the disruption of sphingolipid biosynthesis in vervet monkeys by fumonisins in culture material added to their diet can effectively be monitored in the serum as an elevation of the sphinganine:sphingosine ratio.

Liquid chromatographic determination of fumonisins B1, B2, and B3 in corn: AOAC-IUPAC Collaborative Study.

A liquid chromatographic (LC) method for simultaneous determination of fumonisins B1 (FB1), B2 (FB2), and B3 (FB3) in corn was subjected to a collaborative study involving 12 participants from 10 countries, in which the accuracy and reproducibility characteristics of the method were established. Mean analyte recoveries from corn ranged from 81.1 to 84.2% for FB1 (at a spiking range of 500 to 8000 ng/g), from 75.9 to 81.9% for FB2 (at a spiking range of 200 to 3200 ng/g), and from 75.8 to 86.8% for FB3 (at a spiking range of 100 to 1600 ng/g). The valid data were statistically evaluated after exclusion of outliers. Relative standard deviations for within-laboratory repeatability ranged from 5.8 to 13.2% for FB1, from 7.2 to 17.5% for FB2, and from 8.0 to 17.2% for FB3. Relative standard deviations for between-laboratory reproducibility varied from 13.9 to 22.2% for FB1, from 15.8 to 26.7% for FB2, and from 19.5 to 24.9% for FB3. HORRAT ratios, calculated for the individual toxin analogues, ranged from 0.75 to 1.73. The LC method for determination of fumonisins B1, B2, and B3 in corn (at concentrations of 800-12800 ng total fumonisins/g) has been adopted by AOAC INTERNATIONAL.

Effects of temperature and incubation period on production of fumonisin B1 by Fusarium moniliforme.

The kinetics of the production of fumonisin B1 (FB1) by Fusarium moniliforme MRC 826 in corn cultures was investigated as a function of fungal growth at various incubation temperatures. The growth rate of F. moniliforme, as measured by ergosterol concentration, was higher at 25 degrees C than at 20 degrees C, reaching a stationary phase after 4 to 6 weeks in both cases. FB1 production commenced after 2 weeks during the active growth phase, continued to increase during the stationary phase, and decreased after 13 weeks. The overall maximal yield of FB1 (17.9 g/kg, dry weight) was obtained in corn cultures incubated at 20 degrees C for 13 weeks, but it was not significantly (P greater than 0.05) higher than the maximum yield (16.5 g/kg, dry weight) obtained at 25 degrees C after 11 weeks. However, a significantly (P less than 0.05) higher mean yield was detected at 25 degrees C (9.5 g/kg, dry weight) than at 20 degrees C (8.7 g/kg, dry weight). Production reached a plateau after 7 weeks of incubation at 25 degrees C or 9 weeks of incubation at 20 degrees C. The maximal production of FB1 at 30 degrees C was very low (0.6 g/kg, dry weight). FB1 was also found to be heat stable, as there was no reduction in the FB1 concentration after boiling culture material of F. moniliforme MRC 826.

Primary liver cancer and aflatoxin intake in Transkei.

Transkei has an intermediate primary liver cancer (PLC) rate that is unusually high for a corn-based subsistence economy. The situation is complicated by a very high esophageal carcinoma incidence rate and known exposure to hepatocarcinogenic N-nitrosamines. A total of 623 cooked food samples were collected from two northeastern and two southern districts over two seasons, in a single-sample grid pattern, for aflatoxin analysis. Aflatoxin B1 was quantitated in 26% of all samples during 1976, resulting in a mean contamination rate of 0.65 microgram/kg wet food. Mean contamination during 1977 was 0.66 microgram/kg, and 24% of samples were positive. Thus for both years a mean aflatoxin B1 intake of 16.5 ng/kg body weight per day was calculated. When the data were plotted on our published PLC incidence-aflatoxin intake graph constructed with standardized data from four countries, Transkei was well within the 95% confidence limits. Individual district comparisons are handicapped by as yet inadequate PLC incidence data, but rank order correlations (rs) between aflatoxin intakes and the PLC incidence in goldminers originating from the study districts were significant (P less than .05). Corn was the basis of 95% of the meals and was also the main source of contamination. Six of 23 samples of home-brewed corn beer were positive for aflatoxin, resulting in an overall contamination of 0.69 microgram/L.

Cancer promoting potential of different strains of Fusarium moniliforme in a short-term cancer initiation/promotion assay.

A short-term cancer initiation/promotion bioassay was established to screen 10 toxic strains of Fusarium moniliforme for their cancer promoting activity in rats. The assay consisted of a four week 'promoting' treatment, effected by incorporating culture material (5%) of each strain into the diet, commencing one week after an initiation treatment with diethylnitrosamine (DEN, 200 mg/kg). The appearance of gamma-glutamyltranspeptidase-positive (GGT+) foci was used as an indication of promoting activity. Three out of 10 strains of F. moniliforme obtained from corn from a high risk area for esophageal cancer in Transkei, southern Africa, had significant cancer promoting activity. A highly significant correlation was found between toxicity expressed as reduction in body weight gain and cancer promoting activity. This finding suggests that the compounds responsible for the hepatotoxicity and hepatocarcinogenicity of F. moniliforme could be identical.

Fusarium moniliforme contamination of maize in oesophageal cancer areas in Transkei.

The prevalence of human oesophageal cytological abnormalities was determined by means of brush biopsy capsules in the adult occupants of each of 12 households in a low, an intermediate and a high oesophageal cancer rate area in Transkei during 1985 and 1986. Mild cellular changes (folic acid deficiency, atypia and mild dysplasia) as well as advanced changes (dysplasia and cancer) occurred more frequently in the occupants of households in high than in intermediate and low oesophageal cancer rate areas. The prevalence of fungi in homegrown maize from the households in each area was determined by plating surface-sterilized kernels on agar. The prevalence of Fusarium moniliforme was significantly higher (P less than 0.01) in maize from cytologically 'affected' households in the high oesophageal cancer rate area than from 'unaffected' households in the low oesophageal cancer rate area during both seasons. These results confirm previous reports on the difference in the prevalence of F. moniliforme in maize from low and high oesophageal cancer rate areas in Transkei. Whereas the previously established correlation was between F. moniliforme and oesophageal cancer rate, the present results provide evidence for an association between this fungus and oesophageal cytological abnormalities in living individuals. These people will be included in further clinical, epidemiological and aetiological studies.

Hepatocellular carcinoma and dietary aflatoxin in Mozambique and Transkei.

Estimations of the incidence of hepatocellular carcinoma (HCC) for the period 1968-74 in the Province of Inhambane, Mozambique, have been calculated and together with rates observed in South Africa among mineworkers from the same Province indicate very high levels of incidence in certain districts of Inhambane. Exceptionally high incidence levels in adolescents and young adults are not sustained at older ages and suggest the existence of a subgroup of highly susceptible individuals. A sharp decline in incidence occurred during the period of study. Concurrently with the studies of incidence, 2183 samples of prepared food were randomly collected from 6 districts of Inhambane as well as from Manhica-Magude, a region of lower HCC incidence to the south. A further 623 samples were taken during 1976-77 in Transkei, much further south, where an even lower incidence had been recorded. The mean aflatoxin dietary intake values for the regions studied were significantly related to HCC rates. Furthermore, data on aflatoxin B1 contamination of prepared food from 5 different countries showed overall a highly significant relationship with crude HCC rates. In view of the evidence that chronic hepatitis B virus (HBV) infection may be a prerequisite for the development of virtually all cases of HCC and given the merely moderate prevalence of carrier status that has been observed in some high incidence regions, it is likely that an interaction between HBV and aflatoxin is responsible for the exceptionally high rates evident in parts of Africa and Asia. Various indications from Mozambique suggest that aflatoxin may have a late stage effect on the development of HCC. This points to avenues for intervention that could be more rapidly implemented than with vaccination alone.

Dietary wheaten bran in baboons: long-term effect on the morphology of the digestive tract and aorta, and on tissue mineral concentrations.

Two groups of 13 young baboons, each consisting of 8 males and 8 females, were fed on either high- or low-bran diets (based on wheat of either high or low extraction rate) for a period of 26 months. All animals grew well and remained in good condition throughout. Male (but not female) baboons on the high-bran diet had lower (p less than 0.05) concentrations of zinc in serum and bone, despite a low phytate: zinc molar ratio and a high intake of zinc. Particle-induced X-ray emission analysis showed there to be lower concentrations of calcium, copper, zinc, sulphur, potassium and nickel in the livers of baboons on the high-bran diet (P less than 0.005). Baboons on the low-bran diet passed smaller quantities of softer faeces, they had fewer nodules of lymphoid tissue in the distal portion of the colon (P less than 0.05), and within mucosal microherniations of their ileo-caecal valves the epithelial cells showed a greater tendency to squamous transformation (P less than 0.05). Mucosal microherniations of the ileo-caecal valves tended to be more frequent and larger in size (P less than 0.05) in animals of the low-bran group. Morphometrical studies did not reveal any differences in the general structural development of the digestive tract. The high-bran diet had no effect on serum cholesterol concentrations, nor on the incidence or severity of atherosclerotic lesions of the aorta.

Serum ferritin and mean corpuscular volume as predictors of bone marrow iron stores.

The prognostic value of serum ferritin (SF) as a predictor of bone marrow iron stores (BMS) was evaluated in an attempt to use SF and other readily available clinical and laboratory parameters in the more accurate prediction of BMS without bone marrow aspiration. The results of routine haematological studies of 1000 patients referred for a bone marrow examination as part of their diagnostic evaluation at Tygerberg Hospital were statistically analysed. Significant associations were found. The CHAID programme of automatic interaction detection as developed by Kass was used to determine the optimum classification. A statistical model in the form of a dendrogram has been computed for this hospital population and this laboratory for future predictions of BMS using only the values for SF and mean corpuscular volume. The predictions were statistically very highly significant, especially in patients with iron deficiencies.

The admission of the elderly to places of care: a socio-psychiatric community survey.

Elderly persons over the age of 60 who were admitted for psychiatric care were compared with a random sample of persons living in the same community in respect of psychiatric, medical and socio-economic variables. The group admitted to hospital for psychiatric reasons was very similar to the latter in general characteristics. However, those people admitted to old age homes, which are the other major resource of psychiatric illness, constitute a distinctly separate population, being older and having considerably more physical illnesses and socio-economic problems. Factors predicting admission to an old age home were largely irremediable age-related conditions (dementia, physical infirmity, etc.), but there were also adverse social circumstances, including marked isolation and a lack of social and emotional support. A comparative analysis of the many psychiatric, social and medical factors is presented, with particular reference to reasons for referral and prevention of admission.

Cholesterol-ascorbic acid interactions in guinea pig liver homogenates, and in the in situ perfused liver: relationship between ascorbic acid status and cholesterol and bile acid synthesis from mevalonate.

Cell free homogenates, and in situ perfused livers were used to study cholesterol synthesis and catabolism in control and latently scorbutic guinea pigs. In the homogenate studies, cholesterol synthesis from (2-14C) mevalonate was significantly depressed in latently scorbutic guinea pigs when compared to controls (0.02 less than p less than 0.05). Synthesis of cholesterol from (1-14C) acetate and n (1-14C) octanoate was minimal in guinea pigs. In the in situ liver perfusions, the synthesis of cholesterol from (2-14C) mevalonate was 40% lower in latently scorbutic guinea pigs than in control animals (p less than 0.005). The synthesis of cholesterol from (1-41C) acetate did not show the same clear cut effect. Bile acid production in the in situ perfused liver was unexpectedly higher in the latently scorbutic animals than in control animals (p = 0.005), although the incorporation of the label from (2-14C) mevalonate into the bile acids from the two groups was not significantly different. This finding led to the latently scorbutic group having a far lower specific activity in the bile acid fraction than the replete group.

Size at birth of infants born at term to coloured primiparas in Cape Town.

An in-depth study was made on the size and body proportions of infants born at term to Coloured primiparas in Cape Town. The infants were lighter and shorter than has been found in most comparative studies, but had some sparing of head growth with normal weight/length proportions. This report provides a standard which will facilitate further studies of fetal growth determinants in this community.

[Small-for-age babies: etiological factors in the Cape colored population]. / Klein-vir-datum babas: etiologiese faktore by die Kaapse kleurling-bevolking.

One hundred and thirty-one small-for-dates Cape Coloured newborns and their mothers were compared with 81 appropriately grown Cape Coloured newborns and their mothers in order to determine aetiological factors in intra-uterine growth retardation. The mothers of the small-for-dates babies differed from the control mothers in the following respects: their postpartum weight and weight for height were less, their placentas were lighter, their incomes were lower, they smoked more heavily, attended antenatal clinics less frequently, and their social conditions, educational status, dietary intake, weight gain during pregnancy and attitude towards pregnancy were inferior. Their previous babies weighed less than the previous babies of control mothers. The study mothers, however, had higher total serum protein, serum albumin and haematocrit levels, which can probably be explained by a smaller plasma volume expansion. The VDRL test on cord blood was more often positive in study babies, but the mean cord blood IgM level did not differ in study and control babies. It is concluded that socio-economic factors play a major role in the aetiology of small-for-dates babies in the Cape Coloured population. Controlled trials of dietary supplementation seem to be indicated in high-risk mothers in this population group.

Maternal nutrition and the duration of pregnancy.

Maternal weight assessed at the start of the last trimester was found to be a determinant of the duration of pregnancy in 1022 women. It is suggested that maternal nutrition influences birth weight by altering both the rate of intra-uterine growth and the gestational period.