Oxidative stress and potential biomarkers in tomato seedlings subjected to soil lead contamination

Oxidative stress and defense response in leaves of tomato seedlings exposed to extraneous lead (Pb) at 0–500 mg kg⁻¹ soil for nearly 2 months were investigated. Superoxide radical (O₂⁻) was quantified by electron paramagnetic resonance (EPR). Results showed that levels of O₂⁻, malondialdehyde (MDA), carbonyl group and superoxide dismutase (SOD) increased with the increase of bioavailable Pb. The O₂⁻ level was well correlated with MDA, carbonyl groups and SOD activities, suggesting that O₂⁻ might be responsible for them. Intensities in two bands of SOD isoenzymes increased along with added Pb in treatments against control, implying that multigenic expression in SOD enzymes were activated to counteract O₂⁻ stress. Heat shock protein 70 (HSP70) was induced sensitively by soil Pb, which was to alleviate oxidative damage (i.e. increased carbonyl groups). The overall results indicated that HSP70 and O₂⁻ were the most sensitive parameters and the combination of them might be potential biomarkers of soil Pb contamination in tomato seedlings.     

Stress response and potential biomarkers in spinach (Spinacia oleracea L.) seedlings exposed to soil lead

Oxidative stress and biochemical responses of spinach seedlings to soil Pb stress were investigated by pot experiments. The seedlings were exposed to 0-500 mg kg⁻¹ extraneous Pb. After 30 days of germination, production of O₂⁻, HSP 70, HSP 60, superoxide dismutase (SOD) activities, carbonyl groups and lipid peroxidation was significantly induced by soil Pb. After 50 days, HSP 70 and HSP 60 decreased, and HSP 60 was significantly inhibited at 500 mg kg⁻¹. The results indicated that Pb probably induced oxidative stress and proteotoxicity to the seedlings through O₂⁻ accumulation, and that SOD, HSP 70 and HSP 60 were important defense mechanisms to alleviate the oxidative stress. It is found that O₂⁻, HSP 70 and HSP 60 were the most sensitive parameters and had the potential to act as biomarkers for early warning of soil Pb contamination. Concentrations of soil Pb, exposing time and combination of multiple parameters should be also taken into consideration when assessing soil Pb pollution by these biomarkers.     

Stimulation of oxidant production in alveolar macrophages by pollutant and latex particles

Air pollutant dusts as well as chemically defined particles were examined for their activating effect on oxidant production (O₂^? and H₂O₂) in guinea pig alveolar macrophages (AM). Oxidant production was measured as chemiluminescence of albumin-bound luminol. All particles examined stimulated the AM in a dose-dependent manner to different maximal levels of oxidant production. Amphibole asbestos samples were the most active of all agents studied. Various immune reactants as well as silica, metal-oxide-coated fly ash, polymethyl methacrylate beads, and chrysotile asbestos had intermediate activity, while fugitive dusts, polybead carboxylate microspheres, glass and latex beads, uncoated fly ash, and fiberglass had the lowest activity. In addition to direct stimulatory action on AM, particles also lowered the subsequent responsiveness of the cells to the bacterial peptide stimulant, N-formylmethionyl phenylalanine. This effect was only partially due to the cytotoxicity of the particles. While some relationship appeared to exist between stimulatory activity and cytotoxicity of the particles, the exact role of O₂^? in mediating cytotoxic effects is still open to question. Oxidant production in AM is a parameter which may be important in determining the pathological effects of particles as well as of materials adsorbed to their surfaces.     

A trial on the quantitative risk assessment of man-made mineral fibers by the rat intraperitoneal administration assay using the JFM standard fibrous samples

We tried to evaluate the carcinogenic risk of man-made mineral fiber based on the mesothelioma incidence in female F344 rats after intraperitoneal administration. Rats (female F344/ Nslc, 5-week-old, n=330) were observed for 2 years after the intraperitoneal administration of 5 to 20 mg of 9 types of the JFM (Japan Fibrous Material Research Association) standard fiber samples (glass wool, rock wool, micro fiber glass, three types of refractory fiber, potassium titanate whisker, silicon carbide whisker, titanium oxide whisker), wollastonite (natural fiber) and UICC chrysotile B. All rats administered 10 mg of silicon carbide whisker had developed peritoneal mesothelioma within a year. The cumulative incidence of peritoneal mesothelioma at the end of the experiment was 85% for 10 mg UICC chrysotile B, 77% for 10 mg of potassium titanate whisker, 70% for 5 mg of silicon carbide whisker, 20% for 5 mg of potassium titanate whisker, 20% for 20 mg of refractory fiber 2 and 10% for 20 mg of refractory fiber 1. Carcinogenicity was estimated 2.4 times for silicon carbide whisker and 0.23 for potassium titanate whisker in comparison with UICC chrysotile B. It has been well documented from several experimental studies that man-made fibers are safer than asbestos because of the different durability in the lung. Present results consistently suggest that man-made fibers with high durability have similar or higher risk as carcinogen than asbestos.     

Products of oxidized L-Ascorbic Acid damage acellular DNA

Objective

L-ascorbic acid can be pro-oxidant and anti-oxidant in different reaction. This study aims to test the effects about products of oxidized L-Ascorbic Acid on acellular DNA.

Measurement

Acellular DNA, nuclear DNA fixed on slides, are used in our experiment. There are four groups and one negative. Negative control is sham-treated with buffer(pH 7.2 and AA/H₂O₂/fenton free). Experimental groups are treated separately with 0.06 mM L-ascorbic acid (AA) alone(exposed in air), 0.06 mM L-ascorbic acid (AA) alone(no exposure in air), 1.2 mM hydrogen peroxide (H₂O₂) alone, and a mixture of final concentration of 0.03 mM L-ascorbic acid and 0. 6 mM hydrogen peroxide (AA+ H₂O₂). Each experimental group consists of 4 slides and each slide is treated for 4 hours at 4 °C in a dark place. The DNA damage is quantified by alkaline Comet Assay. The comet images are analysed by Comet Al.0 software. Differences among groups are compared with SPSS 11.0.

Results

DNA singlestrand breakage is found to be treatment-dependent in the following sequence: AA+ H₂O₂> AA(oxidized) > H₂O₂, AA(without oxidization) and Control.

Conclusion

Acellular DNA can tolerate the low concentration H₂O₂, but is sensitive to free radical. The results indicate that AA expose in air and mixture of AA and H₂O₂ can produce ·OH and L-dehydroascorbate (DHA), ·OH can damage DNA.     

Reproducibility of basal and induced DNA single-strand breaks detected by the single-cell gel electrophoresis assay in human peripheral mononuclear leukocytes

The aim of the reported study was to investigate the reproducibility of the single-cell gel electrophoresis (SCGE) assay in the determination of DNA single-strand breaks (SSBs) and to estimate the statistical requirements when the SCGE assay is used for the detection of genotoxicity in humans. In human peripheral mononuclear leukocytes (PMLs), we repeatedly measured the rate of SSBs after in vitro incubation of cells for 1 h at 4°C in phosphate buffered saline (PBS, basal) or 10 M or 50 M H₂O₂ (induced). Intra-assay variation was determined from cryopreserved PMLs of a single donor. To assess intrasubject and intersubject variation, PMLs of ten healthy, nonsmoking subjects (aged 19–37 years) were tested 5–9 times. Cryopreserved cells revealed a mean coefficient of variation of 18% (PBS) and 7%–9% (H₂O₂). There were statistically significant differences between individuals in the rate of SSBs after incubation in PBS (P < 0.01), 10 M H₂O₂ (P < 0.001), and 50 M H₂O₂ (P < 0.001). The range of interindividual variability was 26% for basal and 12%–13% for induced SSBs, and the coefficient of intraindividual variation was 18%–72% (PBS) and 7%–23% (H₂O₂). Neither basal nor induced rates of DNA damage were related to gender or age. Estimates of the minimum detectable effects were based on these observed sources of variability (power 90%, level of significance 5%, assumed sample size 50). With two different groups, a difference of 31% in basal SSBs or 12% in induced SSBs would be detectable. Repeated measurement within one group could detect a difference of 26% in basal and 9% in induced SSBs. In summary, the SCGE assay appears to be suitable for the detection of single-strand breaks, e.g., in biomonitoring or environmental medicine, and the statistical requirements could be derived from our analysis of the sources of variability.This work is part of a thesis by A. Kästner for a medical doctorate at the University of Hamburg.     

Changes in low molecular weight DNA fragmentation in white blood cells of workers highly exposed to asbestos

Objective: The aim of this study was to determine low molecular weight (LMW)-DNA fragmentation changes after white blood cell (WBC) incubation in lysis buffer followed by constant-field gel electrophoresis (CFGE). WBCs were isolated from blood samples of workers highly exposed to asbestos fibres at the workplace in Germany, and were compared with those from healthy adults. This study was conducted parallel to the study presented in our preceding paper (Marczynski et al. 2000b) in which we described significant increases in the levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG) adducts in the DNA of white blood cells from the same highly exposed workers relative to the levels found in the control group in all three study years (1994 to 1997). Method: We found that 15-h incubation in lysis buffer of WBCs embedded in agarose-plugs from healthy control donors with 2% SDS, proteinase K and Na₂-EDTA at 42 °C followed by 0.5 h at 4 °C produced a characteristic DNA fragmentation pattern below 23 kbp using CFGE. Results: In the 1st year of the study (1994–1995) changes were found in LMW-DNA fragmentation in 54.8% of the asbestos workers studied, compared with the DNA fragmentation pattern of controls. Interestingly, in the 2nd year of the study (1995–1996) changes in DNA fragmentation were found in only 39.9% of exposed subjects. In the 3rd year of the study (1996–1997) the highest number of workers exposed to asbestos (67.3%) with changes in the LMW-DNA fragmentation pattern was found. The Chi-square test for each year of the study revealed significant changes (P < 0.001). These changes may be due to the presence of hydrogen peroxide (H₂O₂), as has been shown in vitro. It is likely that a Fenton reaction involving the heterolytic reduction of H₂O₂ by traces of reduced transition metals such as Fe²⁺ and Cu⁺ is involved in the fragmentation of DNA. No difference was found in the changes in DNA fragmentation between asbestos-exposed subjects with and without benign asbestos-associated diseases (asbestosis, asbestos-associated pleural plaques). Significant correlations were not found after analysis of the changes in DNA fragmentation in relation to different possible occupational and non-occupational confounding factors, such as the duration of asbestos exposure, the latency period, estimated cumulative fibrous dust dose (“fibre-years”), and non-occupational confounding factors, such as age, smoking status, acute febrile infections, the intake of medicines, aspirin, Ca²⁺, Mg²⁺ and/or hormones, the intake of vitamins, and cases of cancer. Conclusions: Our data confirm that oxidative stress occurs in the WBCs of workers highly exposed to asbestos fibres, thus supporting the hypothesis that asbestos fibres damage cells through an oxidative mechanism. Oxidative stress and oxidative DNA damage may be induced by long-term exposure to asbestos. The new insights into the oxidative effects of asbestos fibres are of great importance because they provide a way forward for new preventive strategies. Preventive and therapeutic approaches using antioxidants should be taken into consideration. Received: 2 October 2000 / Accepted: 27 January 2001     

Anti-proliferative and pro-apoptotic activities of hydroxytyrosol on different tumour cells: the role of extracellular production of hydrogen peroxide

Purpose

Several recently published data suggest that the anti-proliferative and pro-apoptotic properties of hydroxytyrosol [3,4-dihydroxyphenyl ethanol (3,4-DHPEA)] on HL60 cells may be mediated by the accumulation of hydrogen peroxide (H₂O₂) in the culture medium. The aim of this study was to clarify the role played by H₂O₂ in the chemopreventive activities of 3,4-DHPEA on breast (MDA and MCF-7), prostate (LNCap and PC3) and colon (SW480 and HCT116) cancer cell lines and to investigate the effects of cell culture medium components and the possible mechanisms at the basis of the H₂O₂-producing properties of 3,4-DHPEA.

Methods

The proliferation was measured by the MTT assay and the apoptosis by both fluorescence microscopy and flow cytometry. The concentration of H₂O₂ in the culture medium was measured by the ferrous ion oxidation–xylenol orange method.

Results

It was found that the H₂O₂-inducing ability of 3,4-DHPEA is completely prevented by pyruvate and that the exposure of cells to conditions not supporting the H₂O₂ accumulation (addition of either catalase or pyruvate to the culture medium) inhibited the anti-proliferative effect of 3,4-DHPEA. Accordingly, the sensitivity of the different cell lines to the anti-proliferative effect of 3,4-DHPEA was inversely correlated with their ability to remove H₂O₂ from the culture medium. With regard to the mechanism by which 3,4-DHPEA causes the H₂O₂ accumulation, it was found that superoxide dismutase increased the H₂O₂ production while tyrosinase, slightly acidic pH (6,8) and absence of oxygen (O₂) completely prevented this activity. In addition, different transition metal-chelating compounds did not modify the H₂O₂-producing activity of 3,4-DHPEA.

Conclusions

The pro-oxidant activity of 3,4-DHPEA deeply influences its ‘in vitro’ chemopreventive activities. The main initiation step in the H₂O₂-producing activity is the auto-oxidation of 3,4-DHPEA by O₂ with the formation of the semiquinone, superoxide ions (O₂ ^?) and 2H⁺.     

Smoking-induced increase in urinary cadmium levels among Japanese women

Objective: To examine if cigarette smoking will induce elevation in cadmium (Cd) in urine. Methods: Information on smoking habits, and urinary levels of cadmium (Cd-U), ₁-microglobulin (₁-MG), ₂-microglobulin (₂-MG), creatinine (CR or cr), and urine specific gravity (SG or sg) was cited from a combination of three previously established databases on adult Japanese women. After exclusion of those with unclear answers on smoking habits (412 cases), the combination (12,846 cases) gave 11,092, 1420 and 334 cases of never, current and former smokers, respectively, for present statistical analyses. Results: Multiple regression analyses taking Cd-U as a dependent variable and 11 regions of urine collection, age and smoking habits as independent variables showed that age and regions were powerful confounders in the analysis for the effects of smoking on Cd-U. To exclude the confounding effects, current and former smokers were paired with age- and region-matched never smoking controls in subsequent analyses. In addition, former smokers were paired with age- and region-matched current smokers. The comparison of the paired cases showed that Cd-U for current smokers was significantly higher than that for never smokers. The levels for former smokers were however not higher than the levels for never smokers. When classified by the number of cigarettes consumed per day, Cd-U for current smokers increased dependently to the number of cigarettes (about 0.09 g/cigarette/day) with leveling off at 15 or more cigarettes. There was a subtle cigarette dose-dependent increase in ₁-MG, but the increase was insignificant in case of ₂-MG. Estimation of the amount of Cd absorbed due to cigarette smoking followed by comparison with the increase in Cd-U suggested that almost all Cd absorbed will be excreted into urine. Conclusions: Among currently smoking Japanese women, cadmium in urine increased in a manner dependent to the number of cigarettes consumed daily. Thus, smoking is a confounder of Cd-U evaluation even among the population with relatively high dietary Cd burden.     

Linking oxidative events to inflammatory and adaptive gene expression induced by exposure to an organic particulate matter component

Background: Toxicological studies have correlated inflammatory effects of diesel exhaust particles (DEP) with its organic constituents, such as the organic electrophile 1,2-naphthoquinone (1,2-NQ).Objective: To elucidate the mechanisms involved in 1,2-NQ–induced inflammatory responses, we examined the role of oxidant stress in 1,2-NQ–induced expression of inflammatory and adaptive genes in a human airway epithelial cell line.Methods: We measured cytosolic redox status and hydrogen peroxide (H₂O₂) in living cells using the genetically encoded green fluorescent protein (GFP)-based fluorescent indicators roGFP2 and HyPer, respectively. Expression of interleukin-8 (IL-8), cyclooxygenase-2 (COX-2), and heme oxygenase-1 (HO-1) mRNA was measured in BEAS-2B cells exposed to 1,2-NQ for 1–4 hr. Catalase overexpression and metabolic inhibitors were used to determine the role of redox changes and H₂O₂ in 1,2-NQ–induced gene expression.Results: Cells expressing roGFP2 and HyPer showed a rapid loss of redox potential and an increase in H₂O₂ of mitochondrial origin following exposure to 1,2-NQ. Overexpression of catalase diminished the H₂O₂-dependent signal but not the 1,2-NQ–induced loss of reducing potential. Catalase overexpression and inhibitors of mitochondrial respiration diminished elevations in IL-8 and COX-2 induced by exposure to 1,2-NQ, but potentiated HO-1 mRNA levels in BEAS cells.Conclusion: These data show that 1,2-NQ exposure induces mitochondrial production of H₂O₂ that mediates the expression of inflammatory genes, but not the concurrent loss of reducing redox potential in BEAS cells. 1,2-NQ exposure also causes marked expression of HO-1 that appears to be enhanced by suppression of H₂O₂. These findings shed light into the oxidant-dependent events that underlie cellular responses to environmental electrophiles.     

Consumption of fructose- but not glucose-sweetened beverages for 10 weeks increases circulating concentrations of uric acid, retinol binding protein-4, and gamma-glutamyl transferase activity in overweight/obese humans

Background

Down syndrome (DS) neurons are more susceptible to oxidative stress and previous studies have shown that vitamin E was able to reduce oxidative stress and improve DS neurons' viability. Therefore, this study was done to investigate the protective role of ??-tocotrienol (??T3) in DS neurons from hydrogen peroxide (H₂O₂) -induced oxidative stress. The pro-apoptosis tendency of ??T3 was compared to ??-tocopherol (??T) in non-stress condition as well.

Methods

Primary culture of DS and euploid neurons were divided into six groups of treatment: control, H₂O₂, ??T3 pre-treatment with H₂O₂, ??T3 only, ??T pre-treatment with H₂O₂ and ??T only. The treatments were assessed by MTS assay and apoptosis assay by single-stranded DNA (ssDNA) apoptosis ELISA assay, Hoechst and Neu-N immunofluorescence staining. The cellular uptake of ??T3 and ??T was determined by HPLC while protein expressions were determined by Western blot. Comparison between groups was made by the Student's t test, one-way ANOVA and Bonferroni adjustment as well as two-way ANOVA for multiple comparisons.

Results

One day incubation of ??T3 was able to reduced apoptosis of DS neurons by 10%, however ??T3 was cytotoxic at longer incubation period (14 days) and at concentrations ?? 100 ??M. Pre-treatment of ??T and ??T3 only attenuate apoptosis and increase cell viability in H₂O₂-treated DS and euploid neurons by 10% in which the effects were minimal to maintain most of the DS cells' morphology. ??T3 act as a free radical scavenger by reducing ROS generated by H₂O₂. In untreated controls, DS neurons showed lower Bcl-2/Bax ratio and p53 expression compared to normal neurons, while cPKC and PKC-?? expressions were higher in DS neurons. On the other hand, pre-treatment of ??T3 in H₂O₂-treated DS neurons have reduced Bcl-2/Bax ratio, which was not shown in euploid neurons. This suggests that pre-treatment of ??T3 did not promote DS cell survival. Meanwhile ??T3 and ??T treatments without H₂O₂ as well as pre-treatment of ??T3 and ??T induced changes in cPKC and PKC-?? expression in DS neurons suggesting interaction of ??T3 and ??T with PKC activity.

Conclusion

Our study suggests that ??T3 pre-treatment are not sufficient to protect DS neurons from H₂O₂-induced oxidative assault, instead induced the apoptosis process.     

Comparative epidemiology of men exposed to asbestos and man-made mineral fibers

Comparative analyses are presented of selected studies of long-term reactions to occupational exposures to asbestos and man-made mineral fibers (MMMF), with emphasis on studies with dose-response information and long enough period of follow-up to observe lung cancer excess, if it occurred. Uniform dose estimates based on average number of fibers per milliliter were derived and tabulated with the corresponding standard mortality (or morbidity) ratio (SMR), crude probability for each unfavorable outcome, and the likelihood that at least as many deaths would have occurred as a result of the expected numbers under Poisson assumptions. A dose-response relationship was said to have been indicated when the crude probability increased monotonically with dose and/or the Poisson probability decreased and reached a value of less than 0.05. Some arbitrary assumptions had to be made in estimation of the dose, and they may need to be corrected. Gravimetric dose estimates may have given different results. Studies selected for analysis included Quebec asbestos miners and asbestos cement workers exposed to asbestos, and pooled U.S. and European studies of MMMF workers, as well as a sample of cigarette-smoking fiberglass workers whose X-ray films were evaluated for fine nodular or irregular opacities. The lowest dose capable of showing either a statistically significant excess (single point criterion--SP) or the median dose in an apparent dose-response relationship with cause of death or radiological results is tabulated. Radiological changes show a dose-response relationship for all types, with a median dose for asbestos of 2.8 fibers/ml. For fiberglass workers, the median dose of electron-microscopically detected fibers was two orders of magnitude less. For asbestos SP, exposures of 1.4 to 22 fibers/ml were associated with increased lung cancer, while for mineral wool, the minimal level with significant SP increase in lung cancer was an order of magnitude less. Based on fiber or particle counts, man-made mineral fibers appear to be more potent than asbestos with regard to chronic pulmonary disease.     

Activity of glutamate decarboxylase in the brain of rats exposed to carbon disulfide

Summary The activity of glutamate decarboxylase in the brain of white rats exposed to CS₂ was measured. The lower activity of the enzyme in rats intoxicated with CS₂ was parallel to the decreased level of -aminobutyric acid. Pyridoxal phosphate did not prevent inhibition of the enzyme activity. It is suggested that the inhibition of the activity of glutamate decarboxylase by CS₂ is responsible for lower level of -aminobutyric acid in the brain of rats intoxicated with CS₂.     

In vivo and in vitro evidences that carotenoids could modulate the neutrophil respiratory burst during dietary manipulation

Summary Background The primary role of polymorphonuclear neutrophils (PMNs) is to destroy pathogenic microorganisms after phagocytosis by producing reactive oxygen species (ROS) and toxic molecules. However, PMNs produce sufficient amounts of ROS during an oxidative burst to be autotoxic and detrimental to their own functions and to possibly cause DNA damage, protein and lipid oxidation and cell membrane destructuration. Objective The aim of this study was to investigate in vivo the role of the antioxidant capacities of carotenoids in modulating ROS content in PMNs during oxidative burst. Moreover to investigate the direct or indirect effect of carotenoids, the modification of PMN ROS content was explored after in vitro supplementation with -carotene or lycopene, chosen taking account of their vitamin A and no vitamin A precursor effect, respectively. Design In vivo study: Venous blood was collected from 10 healthy male volunteers and ROS production from phorbol myristate acetate (PMA)-stimulated PMNs was determined, by flow cytometry using the fluorescent dye dihydrorhodamine 123, at baseline, after 3 weeks of carotenoid depletion (carotenoid intake limited to 25% of usual intake) and after 5 weeks of carotenoid repletion (30 mg -carotene, 15 mg lycopene and 9mg lutein per day). In vitro study: ROS content in PMA-stimulated PMNs isolated from carotenoid depleted subjects and controls was quantified after an in vitro enrichment with -carotene (1 µmol/L) or lycopene (0.3 µmol/L). Results In vivo carotenoid depletion increased PMN H₂O₂ content after PMA activation by 38% (p < 0.05 vs baseline),while supplementation for 5 weeks restored basal H₂O₂ generation (p < 0.05 vs depletion). Although H₂O₂ measurement in PMNs from non-depleted subjects was not affected by an in vitro supply with -carotene or lycopene, a significant decrease in H₂O₂ content by 78.9 % and 81.2%, respectively, was observed in PMNs from carotenoid depleted subjects (p < 0.01 vs depleted control subjects). Conclusions The carotenoid ROS quenching capacities control both in vivo and in vitro the PMNs ROS generation and probably protect these cells against DNA, membrane lipid and protein damages during oxidative burst. Moreover, these effects appear independent from the metabolic conversion of carotenoids to vitamin A.     

Aluminium induced oxidative stress and DNA damage in root cells of Allium cepa L

Aluminium (Al) was evaluated for induction of oxidative stress and DNA damage employing the growing roots of Allium cepa L. as the assay system. Intact roots of A. cepa were treated with different concentrations, 0, 1, 10, 50, 100, or 200 μM of aluminium chloride, at pH 4.5 for 4 h (or 2 h for comet assay) at room temperature, 25±1 °C. Following treatment the parameters investigated in root tissue were Al-uptake, cell death, extra cellular generation of reactive oxygen intermediates (ROI), viz. O₂⁻, H₂O₂ and OH, lipid peroxidation, protein oxidation, activities of antioxidant enzymes namely catalase (CAT), superoxide dismutase (SOD), guaiacol peroxidase (GPX), ascorbate peroxidase (APX); and DNA damage, assessed by comet assay. The findings indicated that Al triggered generation of extra-cellular ROI following a dose-response. Through application of specific enzyme inhibitors it was demonstrated that extra-cellular generation of ROI was primarily due to the activity of cell wall bound NADH-PX. Generation of ROI in root tissue as well as cell death was better correlated to the levels of root Al-uptake rather than to the concentrations of Al in ambient experimental solutions. Induction of lipid peroxidation and protein oxidation by Al were statistically significant. Whereas Al inhibited CAT activity, enhanced SOD, GPX and APX activities significantly; that followed dose-response. Comet assay provided evidence that Al induced DNA damage in a range of concentrations 50–200 μM, which was comparable to that induced by ethylmethane sulfonate (EMS), an alkylating mutagen served as the positive control. The findings provided evidence that Al comparable to biotic stress induced oxidative burst at the cell surface through up- or down-regulation of some of the key enzymes of oxidative metabolism ultimately resulting in oxidative stress leading to DNA damage and cell death in root cells of A. cepa.     

β-carotene bioavailability from differently processed carrot meals in human ileostomy volunteers

Summary. Background: Carotenoids contribute to the beneficial effects of fruits and vegetables consumption; however, the bioavailability of these compounds from fresh or processed foods is not well established. Aim of the study: We evaluated the bioavailability of -carotene (15 mg) from a single meal composed of cooked, pureed carrots and compared it to raw, chopped carrots. Methods: Test meals were given to overnight-fastedileostomy volunteers (n = 8) along with skimmed-milk yogurt containing 40 g of added sunflower oil. Blood and complete ileal effluent samples were collected over a 24 h period. Samples were solvent-extracted and the -carotene content measured by HPLC. Results: Kinetics of excretion of cis and trans -carotene were similar. More -carotene was absorbed from puree as compared to raw carrots. Carotenoid mass-balance calculations indicated that 65.1 ± 7.4% of the -carotene was absorbed from cooked pureed carrot meals, vs. 41.4 ± 7.4 % from raw, chopped carrot meals. Gastrointestinal transit parameters did not differ significantly among the volunteers. As expected, the calculated lag phase was five times longer for raw vs. cooked carrots. Mean t-end, t-1/2 and rate of mass transit resulted in similar values for both raw and cooked carrot meals. A moderate response in carotenoid plasma profile was observed for cooked carrot test meals. Conclusions: Significantly more -carotene was absorbed from meals containing cooked, pureed carrots than from meals containing the raw vegetable. Moderate carotenoid plasma response was detected within 6 h following the administration of cooked processed carotenoid-containing single meal.     

Similar cholesterol-lowering properties of rice bran oil, with varied gamma-oryzanol, in mildly hypercholesterolemic men

Summary Background The cholesterol lowering properties of rice bran oil (RBO) containing differing amounts of non–saponifiable components have not been studied in humans, to our knowledge. Aim of the study To evaluate cholesterol lowering effects of RBO, with low and high amounts of –oryzanol (ferulated plant sterols) in mildly hypercholesterolemic men. Methods Mildly hypercholesterolemic men, 38–64 y, starting cholesterol 4.9–8.4 mmol/l (n = 30), consumed 50 g/d peanut oil (PNO) in vehicles for 2 wks during a run–in period, then, without wash–out, were randomly equilibrated (based on initial level of cholesterol) into two groups to consume 50 g/d RBO low (0.05 g/d) or high (0.8 g/d) –oryzanol for 4 wks, in a randomized, controlled, parallel design study. Subjects were free–living and consumed habitual diets with some restrictions. Plasma concentrations of total, LDL–,HDL–cholesterol and triacylglycerol were measured at base line and after 2, 4, and 6 wks. Results The two RBO types were not significantly different with respect to effects on various cholesterol parameters, at 2 and 4 wks, including total cholesterol, LDL–, HDL– and LDL/HDL cholesterol ratio. Low and high –oryzanolcontaining RBO feeding for 4 wks lowered total plasma cholesterol (6.3 %), LDL–C (10.5 %) and the LDL–C/HDL–C ratio (18.9 %). Conclusions RBO supplementation at ca. 50% total fat intake improved lipoprotein pattern in mildly hypercholesterolemic men. Methylated sterols in –oryzanol are thought to be largely ineffective at inhibiting dietary cholesterol absorption, but could enhance cholesterol–lowering ability of 4–desmethylsterols. Assuming all ferulated sterols become de–ferulated in the gut, low and high –oryzanolcontaining RBOs provided intestinal loads of 453 and 740 mg/d free 4–desmethylsterols, respectively. This intestinal load of 453–740 mg/d of efficacious free plant sterol equivalents had identical effects on lipoproteins.*This work appeared in part in FASEB J. 14: A250, 2000     

Mechanism for bone disease found in inhabitants environmentally exposed to cadmium: decreased serum 1α, 25-dihydroxyvitamin D level

Summary To investigate the mechanism for bone disease caused by exposure to cadmium serum samples were collected from 5 itai-itai disease patients, 36 Cd-exposed residents with renal tubular damage and 17 non-exposed individuals and analyzed for 1,25-dihydroxyvitamin D[1, 25(OH)₂D], parathyroid hormone, ₂-microglobulin, calcium and inorganic phosphorus. Measurement of percentage tubular reabsorption of phosphate (%TRP) were performed only on the Cd-exposed subjects. Serum 1, 25(OH)₂D], levels were lower in itai-itai disease patients and cadmium-exposed subjects with renal damage than in non-exposed subjects. Parathyroid hormone and ₂-microglobulin concentrations in serum were higher in the Cd-exposed subjects. Decrease in serum 1,25(OH)₂D levels were closely related to serum concentrations of parathyroid hormone, ₂-microglobulin and %TRP. This study suggests that cadmium-induced bone effects were mainly due to a disturbance in vitamin D and parathyroid hormone metabolism, which was caused by the cadmium-induced kidney damage.     

Bromsulphalein (BSP) kinetics in the rat: A new approach in evaluating experimental hepatotoxicity

Summary An animal model for the identification and definition of toxic liver damage, based on the investigation of the BSP metabolism in the rat is proposed.Different hepatotoxins can induce specific functional alteration on the different steps of the BSP hepatobiliary transport, mainly the uptake by hepatocytes and the biliary excretion.Removal curves of BSP from the plasma compartment as well as the biliary secretion were evaluated in rats treated with either -naphthylisothiocyanate (ANIT) for metabolic cholestasis, or with carbon tetrachloride (CCl₄) for fatty infiltration and necrosis of the liver.The data were compared with those obtained with untreated rats and with animals submitted either to complete or incomplete mechanically induced cholestasis.Our results lead to the conclusion that a satisfactory discrimination among different types of liver damage may be obtained when only two plasma parameters of BSP metabolism are considered: the disappearance rate for the early 5 min (K), and 15-min plasma BSP retention (R₁₅).The model is proposed as a suitable tool for the evaluation of experimental hepatotoxicity in living rats giving a characterisation of the functional alteration and a measure of liver impairment.Supported by grants from Consiglio Nazionale delle Ricerche (Roma) and Regione Piemonte (Torino)     

4,4′-Methylenedianiline in hydrolysed serum and urine from a worker exposed to thermal degradation products of methylene diphenyl diisocyanate elastomers

A 45-year-old mechanic employed in blowing hot air (350°–600°C) onto the surface of a polyurethane methylene diphenyl diisocyanate (MDI) conveyer belt developed dyspnoea, rhinoconjunctivitis and fever. The illness was suggestive of an MDI-associated illness, compatible with both immediate hypersensitivity and a complement-mediated immune-complex reaction. In his serum there were specific IgG and IgE antibodies against MDI and other isocyanates, and high values of circulating immune complexes. The patient's blood and urine samples were analysed for the presence of 4,4-methylenedianiline (MDA) in hydrolysed urine and plasma. MDA was derivatized to amides using pentafluoropropionic acid anhydride (PFPA). Gas chromatography-mass spectrometry determinations were made monitoring the (M-20; M = molecular mass) fragments from the MDA-PFPA and the [²H₂]MDA-PFPA derivative. The first urine sample was obtained 22 h and the last sample 114 h after start of exposure. The urine concentrations of MDA were corrected for creatinine. The half-time of MDA was 70–80 h. The first serum sample was obtained 19 h and the last sample 1967 days after the start of exposure. The half-time was 21 days, which suggests the presence of MDI/MDA plasma protein adducts in the exposed worker.