Effect of Ascorbic Acid and Thiamine

Ann.Occup.Hyg.,V ol.51,No.6,pp.563–569,2007ÓThe Author2007.Published by Oxford University Presson behalf of the British Occupational Hygiene Societydoi:10.1093/annhyg/mem036 Effect of Ascorbic Acid and ThiamineSupplementation at Different Concentrations onLead Toxicity in LiverCHUNHONG WANG1*,JIANCHENG LIANG1,CHUNLIAN ZHANG1, YONGYI BI1,XIANGLIN SHI2and QUN SHI11Department of Toxicology,School of Public Health,Wuhan University,DongHu Road115,Wuhan 430071,People’s Republic of China;2Institute for Nutritional Sciences,SIBS,CAS,Shanghai20032, People’s Republic of ChinaReceived16May2007;infinal form11June2007Objective:To investigate the effect of ascorbic acid[vitamin C(VC)]on liver damage param-eters in the lead-exposed mice,when given in combination with thiamine[vitamin B1(VB1)]atdifferent concentrations.Methods:Sixty-six male mice were randomly assigned into11groups(n56).Mice in GroupI were supplied with only the tap water as the drinking water;mice in Group II were providedwith a tap water containing0.2%lead acetate;mice in Group III–XI were given different doseof VC(140,420,1260mg kg21bw)and VB1(10,30,90mg kg21bw)according to333factorialdesign by oral gavages,along with ingestion of0.2%lead acetate.After42test days,DNA dam-age of liver cells was assessed using single-cell gel electrophoresis.The apoptosis rate of livercells was determined byflow cytometry.The activities of superoxide dismutase(SOD)and glu-tathione peroxidase(GSH-Px)in blood and the level of reduced glutathione(GSH)in liver cellswere measured based on individual biochemical reactions.Results:Compared with the Group I,sub-chronic lead ingestion(Group II)resulted in a sig-nificant decrease of Hb,GSH-P X,SOD in blood and GSH level in liver cells;lead exposure in-duced also a significant increase in DNA damage and apoptosis of liver cells(P<0.05).Supplementation with VC and VB1,however,reversed these effects.The best effective combi-nation was VC(420mg kg21bw)and VB1(10mg kg21bw),followed by the combination of VC(420mg kg21bw)and VB1(30mg kg21bw).But no reversion was shown in the combination ofthe highest combination of VC(1260mg kg21)and VB1(90mg kg21).Conclusions:Thesefindings strongly indicated that combination of VC and VB1can lessenthe damage to liver cells from oxidative stress induce by lead,but the antioxidant effects aredependent on their concentrations.Keywords:ascorbic acid;DNA damage;lead;liver;thiamineINTRODUCTIONLead-induced oxidative stress in blood and other soft tissues has been postulated to be one of the possible mechanisms of lead-induced toxic effects(Pande et al.,2001).Disruption of pro-oxidant/antioxidant balance might lead to the tissue injury.It was re-ported that lead increased the level of lipid peroxida-tion(Upasani et al.,2001)and brain thiobarbituric acid-reactive substances and altered the antioxidant defense system(Adanaylo and Oteiza,1999).Similar effects were also reported in the hepatic tissues (Sandhir and Gill,1995).A number of recent studies confirmed the possible involvement of reactive oxy-gen species(ROS)in lead-induced toxicity(Gurer and Ercal,2000).Several antioxidant enzymes and molecules have been used to evaluate lead-induced oxidative damage in animal and human studies.Reduced glutathione (GSH)and glutathione disulfide(GSSG)concentra-tions,as well as modifications in superoxide dismu-tase(SOD)activity are the most frequently used markers in tissues or in blood.*Author to whom correspondence should be addressed.Tel/fax:þ86-27-68758648;e-mail:chwang027@563 by guest on March 7, 2011 Downloaded fromBased on the observation that free radical was gen-erated during the pathogenesis processes induced by lead exposure,it was presumed that supplementation of antioxidants could be an alternative method for chelation therapy (Flora et al.,2003).Specifically,ascorbic acid,the known chelating agent with anti-oxidant features,was widely reported with the capability of protecting cells from oxidative stress (Ramanathan et al.,2002;Patra and Swarup,2004).Besides,additional protective effect of vitamin C (VC)on cell apoptosis was reveled in a recent study (Gruss-Fischer and Fabian,2002).Thiamine,the endogenous –SH containing mole-cule,was recognized as one of the protective agent for lead exposure (Ghazaly,1991).Additional inves-tigations have confirmed that the simultaneous administration of vitamin B 1(VB 1)and VC is more efficient in protecting or treating the experimental lead intoxication than either of them individually (Flora and Tandon,1986;Dhawan et al.,1988).Although the investigations about prevention of lead toxicity utilizing VC and VB 1are fairly well de-fined and previously been considered,reports on the impacts of simultaneous administration with VC and VB 1at different concentrations on lead exposure are novel.Moreover,some authors have raised questions pertaining to the potential risk for side effects of with intake of large doses of vitamins such as VC or tocopherol (Aiguo,2001).In fact,some people in China are intaking vitamin supplement largely.These also deserve deeper investigations.It was pre-sumed that these vitamins might act as pro-oxidant for the introduction of apoptotic cells in the presence of oxygen and the generation of H 2O 2.This study was therefore conducted to investigate whether the combinations of VC and VB 1at various concentra-tions are able to protect the target organs (liver)from lead-associated oxidative stress.MATERIALS AND METHODSAnimals and experimental designAnimal used were Kunming albino male mice (n 566)at the initial age of 5–6weeks,weighting 15–4g.Animals were purchased from the Labora-tory Animal Center of Wuhan University,China.An-imals were accommodated in polypropylene cages in the laboratory animal house,with a 12-h dark/light cycles,at the temperature of 20–25°C and relative humidity of 30–70%.Mice were provided with the standard rodent feeds procured from the Laboratory Animal Center of Wuhan University.Water was sup-plied ad lib.After 7days acclimatization period,mice were randomly assigned into 11groups (n 56).Mice in Group I served as negative control and received tap water alone;mice in Group II served as positive con-trol and received 0.2%lead acetate in drinking water;mice in Group III–XI were given different doses of VC (140,420,1260mg kg À1bw)and VB 1(10,30,90mg kg À1bw)according to 3Â3factorial design by oral gavage daily,along with intaking of the 0.2%lead acetate as shown in Table 1.All experimental animals were under the described treatments for con-tinuous 42days.For each mice,the average water consumption was 5.6ml day À1,so total lead intake was about 470.4mg during experiment.At the end of treatment,blood samples were col-lected in heparinized vials and the liver was removed and kept in a container with the ice jacket for follow-ing analysis.Biochemical analysisActivity of SOD and GSH-Px in blood.The SOD activity in blood was determined using the method modified from the colorimetric NADH–phenazine-methosulphate–nitroblue tetrazolium formazan inhi-bition reaction assay and was expressed in U g À1of hemoglobin (Kakkar et al.,1984).The glutathione peroxidase (GSH-Px)activity in blood erythrocyte hemolysates was measured using the coupled method described by Paglia and Valen-tine (Paglia and Valentine,1967;Hatanaka et al.,2000),with the t -butylhydroperoxide as the sub-strate.The enzyme activity was expressed in interna-tional units per gram of hemoglobin.Assay of GSH content in liver.About 0.2g liver was trimmed in ice-cold saline (0.85%w/v NaCl)and gently homogenized in the ice-cold 1.17%KCl of the 0.10mol l À1phosphate buffer (pH 7.4)at a ra-tio of 10%(w/v),using a glass Potter-type homoge-nizer at 500–800rpm.The homogenates were filtered through a muslin cloth and were centrifuged at 20000g for 30min at 4°C to obtain the supernatants.The GSH was measured in deproteinized superna-tant fractions from liver homogenates,employing 0.04%5,59-dithiobes-(2-nitrobenzoicacid)in 10%Table 1.The dose of lead acetate and vitamin in different groups Group Leadacetate (%)VC(mg kg À1bw)VB 1(mg kg À1bw)I 000II 0.200III 0.214010IV 0.214030V 0.214090VI 0.242010VII 0.242030VIII 0.242090IX 0.2126010X 0.2126030XI0.2126090564 C.Wang et al.by guest on March 7, 2011 Downloaded fromsodium citrate and recording absorption at 412nm on a spectrophotometer (Meng and Bai,2004).Assessment of DNA damageThe single-cell gel electrophoresis assay (SCGE)or comet assay was used for the detection of sin-gle-strand DNA breaks and reparation in individual cells.The method was modified from that reported by Wang et al.,(2006)as follows.Preparation of liver cell suspensions.A liver specimen was cut into pieces with the addition of a phosphate-buffered saline (PBS)solution,followed by a cleaning through a filter.The cellular suspension was then centrifuged at 1000rpm for 10min;the supernatant was discarded and the cell pellets were resuspended in an EP tube.The concentration of sus-pension was adjusted to 106–107cells ml À1for each sample.SCGE assay.Slides were cleaned with acid wash and spread with 40l l of 0.6%agarose,then stood for $5min at room temperature for the gel to set.Onto the first gel layer,a mix of a 20-l l live cell sus-pension and a 80-l l 1.1%low-melting agarose was laid.The slide was then immersed in a cold lysis buffer (2.5mol l À1NaCl,100mmol l À1Na 2EDTA,10mmol l À1Tris,pH 10,1%Triton X-100and 10%dimethyl sulfoxide)for at least 1h at 4°C.At completion of those procedures,slides were arranged on the electrophoresis chamber and fresh prepared electrophoresis buffer (1mmol l À1Na 2EDTA and 300mmol l À1NaOH,pH 12)was added to the cham-ber.After rinsing the slides with PBS for $20min,electrophoresis was performed at 25V ,300mA for 30min.All procedures were performed in the ab-sence of strong light.At the completion of the elec-trophoresis,slides were neutralized by washing with a 0.4-mol l À1Tris–HCl buffer (pH 7.5)for three times,5min each.Slides were then covered with 40l l of 0.1mg ml À1propidium iodide (PI)for the DNA ing a fluorescence microscope (Olympus,Japan)configured with the wavelengths of excitation (510–560nm)and filtration (690nm),100cells from the randomly selected filed were counted and the photograph was taken in each slide.The images were scored for each sample using an image analysis software system (IMI 1.0).Parameters for assessing DNA single-strand breaks included the percentage of cells with tail and tail length by visual estimation.Apoptosis analysisHepatic cells apoptosis were determined with the appearance of the DNA fragments in the apoptotic cells using flow cytometry (FCM).The method used for the detection of the DNA condensation and frag-mentation was modified from the subdiploid DNA peak assay described by Iavicoli et al.,(2001).Ge-netic materials can be bound by the DNA bindingfluorochrome,such as PI and ethidium bromide.Vi-able cells resist the intake of the fluorochromes due to the integration of the cell membrane.Cells stained with these fluorescence indicate the occurrences of the late-stage apoptosis or the appearances of necro-sis.On the DNA histogram figures acquired from the FCM,the diploid DNA peak (G1cells)depressed in apoptotic cells and the subdiploid DNA peak (SD peak)appeared ahead of the G1peak.The ap-pearance of an SD peak represents cells apoptosis.Necrosis cells do not have SD peak,but it exhibits consecutive small DNA fragment curves.Subdiploid DNA peak assay:(i)Aliquot 1$5Â106cells in suspension and centrifuge at 500rpm for 5min.Discard the supernatant;(2)Fix cells in 70%alcohol (À20°C)for 2$24h until analysis;(3)Add PI to a final concentration of 20l g ml À1.Mix well,and let stand at 4°C for 30min in order to allow the dye to equilibrate;(4)Analyze with a flow cytometer with an argon–ion laser tuned to 488nm.Record the light emitted into the red fluores-cence photomultiplier (PMT)using a 620-nm long pass filter in front of the PMT and (5)Analyze the percentage of SD peak which represents the percent-age of apoptotic cells.Statistical analysisThe analyses were performed with the SPSS 11.5software package.The analysis of variance was ap-plied for the comparison of the means of the different treatment groups.Post hoc testing was performed for inter-group comparison applying the least significant difference.Values of P ,0.01and P ,0.05were considered to be significant.RESULTSBiochemistryChronic lead ingestion (Group II)resulted in a sig-nificant decrease in blood Hb,GSH-P X ,SOD and in liver GSH levels after 42days of treatment (Table 2).Supplementation with VC and VB 1,however,re-versed these effects.Moreover,the biochemistry pa-rameters investigated showed no significant differences in Group III,VI and VII with that of the negative control (Group I)(P .0.05).DNA damage of liver cellsAs shown in Table 3and Fig.1A,B,animals in the negative control (Group I)exhibited low but measur-able levels of DNA migration in liver cells assessed by the comet assay.The analysis of the data showed that lead intake increased percentage of cells with tail and tail length.Significant difference was observed in animals in positive control (Group II)after 6weeks of lead exposure in comparison with the neg-ative control (P ,0.01).Combined administrationEffect of ascorbic acid and thiamine supplementation 565by guest on March 7, 2011 Downloaded fromwith VC and VB 1resulted in a significant reduction in the tail length and in the rate of DNA damage,es-pecially in Group VI and VII,in comparison with that of Group II (P ,0.05).From Table 3,we observed the lower values of DNA damage at Group VI.The percentage of liver apoptotic cells was shown in Table 3and Fig.2.In the negative control group (Group I),only about 5%apoptotic cells were de-tected.After lead intake,the percentage of liver apo-ptotic cells (11.9%)in positive control (Group II)was significantly higher than that in Group I (P ,0.01).Except Group XI,the percentage of liver apo-ptotic cells was markedly decreased in all other groups administered with combinations of VC and VB 1,as compared with that of Group II (P ,0.01or P ,0.05).In consistent with the finding in bio-chemistry,the percentage of apoptotic cells in Group VI and VII was also lowest (5.8%,5.2%).The Olive tail length resulted from SCGE in Table 3showed similar reversing effect induced by the sup-plementation of vitamins.Factorial analysis showed that the best effective combination was 420mg kg À1bw VC and 10mg kg À1bw VB 1,the second best was 420mg kg À1bw VC and 30mg kg À1bw VB 1.However,the combination of the highest dose VC (1260mg kg À1)and VB 1(90mg kg À1)had no effect on lead toxicity of oxidative damage (Fig.3).DISCUSSIONLead poisoning has been among the most studied health problems over the years.One of the ways thatTable 2.Changes of blood Hb,GSH-P X ,SOD and liver GSH levels in mice Group Blood GSH in liver (l g/100mg,wet)Hb (g dl À1)GSH-Px (U g À1,Hb)SOD (U mg À1,Hb)I (control)15.47–1.06241.5–24.923.23–1.92233.4–29.4II (lead)11.42–1.09b 193.0–23.9a17.51–2.29b173.1–27.3b III 14.14–0.98d 224.5–25.0c 21.08–2.13d 210.8–21.3d IV 13.14–1.16b,d 216.4–24.619.26–1.98b 200.0–22.0V 12.27–0.85b 207.5–27.4a 18.88–2.06b 191.1–31.9b VI 14.98–1.08d 238.2–25.7d 22.76–2.50d 226.3–29.7d VII 14.20–0.91d 213.2–29.4c 21.96–2.69d 217.4–33.7c VIII 13.72–0.91b,d 220.6–22.420.24–2.31d 205.2–21.2IX 12.75–1.39b 211.3–25.519.54–2.43b 193.2–20.3b X 12.00–1.43b 203.0–22.0b 18.58–1.90b 183.4–27.7b XI11.71–1.38b197.0–24.6b17.88–1.72b181.7–23.3ba P ,0.05orbP ,0.01compared with control group (Group I);cP ,0.05or dP ,0.01compared with only lead-exposed group (Group II).Table 3.The results of comet assay and apoptosis analysis in liver cells of different groups Group Cells with tail (%)Tail length (l m)Olive tail length (l m)Apoptosis rate (%)I (control)15.715.1–8.7 5.0–3.6 4.9–0.9II (lead)67.2b53.4–14.5b18.3–6.1b11.9–1.0b III 28.6b,d 22.6–9.4a,d 8.6–4.3a,d 6.9–0.8a,d IV 38.2b,d 27.5–10.3b,d 10.5–4.9b,d 8.1–0.6b,d V 44.6b,d 34.1–11.5b,d 11.8–5.2b,d 9.7–0.8b,c VI 19.2d 19.0–8.8d 5.9–3.7d 5.2–0.6d VII 21.6a,d 20.8–9.1d 6.7–4.1d 5.8–0.8d VIII 36.4b,d 25.8–9.6b,d 9.4–4.6b,d 7.5–0.9a,d IX 41.0b,d 30.4–10.8b,d 10.8–4.8b,d 8.9–0.9b,c X 48.8b,d 36.6–11.7b,d 13.4–5.5b,d 10.4–0.8b,c XI56.0b,d41.6–13.3b,d15.1–5.8b,d11.1–0.8ba P ,0.05orbP ,0.01compared with control group (Group I);cP ,0.05or dP ,0.01compared with only lead-exposed group (Group II).566 C.Wang et al.by guest on March 7, 2011 Downloaded fromlead exerts its toxic effects is through oxidative stress that may be an important contributor to the negative pathogenesis observed after lead exposure.This study clearly showed that chronic lead inges-tion results in a significant decrease in blood Hb,GSH-P X ,SOD and in liver GSH levels.Under nor-mal conditions,cells possess enzymatic and non-enzymatic defenses to cope with free radicals,such as SOD,GSH-Px,catalase and GSH (Shalana et al.,2005).Oxidative damage,however,may occur when antioxidant potential is decreased and/or when oxidative stress is increased.Some investigators had indicated that lead exposure increased the production of ROS in the liver (Patra et al.,2001;Pande and Flora,2002).Patra reported that lead exposure is in-deed associated with significant increases in lipid peroxide level in the liver.Free radical-induced oxidative damage has been implicated in the pathogenesis of a number of injury and disease states.We have previously found that ROS played a pivotal role in apoptosis of testis cells in lead-exposed mice (Wang et al.,2006).In the pres-ent study,it was exhibited that a significant increase in DNA damage and apoptosis in liver cells occurred via ROS as it played a very important role in apopto-sis induction under both physiological and pathologi-cal conditions.Interestingly,mitochondria were both the source and target of ROS.ROS,whichwasFig.1.DNA damage of liver cells.(A)Liver cell of tail was observed obviously after mice treated lead for 6weeks (Â400).(B)Length of tail cell was decreased significantly at the Group VI (VC 420mg kg À1bw and VB 110mg kg À1bw (Â400).Fig.2.The percentage of liver apoptotic cells (FCM,stained by PI).(A)Control group;(B)only lead-exposed;(C)Group VI (VC 420mg kg À1bw and VB 110mg kg À1bw (Â400)and (D)Group VII (VC 420mg kg À1bw and VB 130mg kg À1bw (Â400).Effect of ascorbic acid and thiamine supplementation 567by guest on March 7, 2011 Downloaded frompredominantly produced in the mitochondria,led to the free radical attack of membrane phospholipids and loss of mitochondrial membrane potential,which caused the intermembrane proteins,such as cyto-chrome c,to be released out of the mitochondria and ultimately triggered caspase-3activation.Caspase-3activation led to DNA breakage,nuclear chromatin condensation and cell apoptosis (Li et al.,2006;Wang et al.,2006).After coadministrated with vitamins,the lead tox-icity was normalized effectively.The levels of GSH-P X ,SOD in blood and GSH in the liver had a significant increase when compared with lead-exposed group (P ,0.01).Namely,free radicals in-duced by lead may oxidize GSH and the vitamin treatment reversed lead-induced decreases in GSH levels.It is well known that ascorbic acid directly re-acts with lipid hydroperoxides and restores the anti-oxidative potency of a -tocopherol.Production of GSH is considered to be the first line of defense against oxidative damage and free radical generation where GSH functions as a scavenger and a cofactor in metabolic detoxification of ROS (Tandon et al.,2003).As discussed previously,ROS played a piv-otal role in apoptosis.So lead-exposed mice co-treated with ascorbic acid could directly reacts with lipid hydroperoxides or/and increase the levels of GSH and then prevented liver destruction from apoptosis.The exact mechanism of thiamine in antagonizing lead toxicity has not been clearly elucidated until now.It might be attributed to the formation of com-plexes between thiamine and lead followed by its excretion.Thiamine also has been found to protect against lead-induced lipid peroxidation in rat liver and kidney (Senapati et al.,2000).It may scavenge O 2 Àand OH directly and thus affect the cellular re-sponse to oxidative stress (Jung and Kim,2003).Anna and Wig ło (2006)reported that thiamine may act as a potent antioxidant as it scavenges free radi-cals such as 2,2-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)radical cation relatively slowly.Its radical-scavenging activity,expressed as the trolox equivalent antioxidant capacity value,is comparable to such natural antioxidants as some carotenoids.The fact that simultaneous supplementation of ascor-bic acid and thiamine ameliorated the liver damage in lead-treated mice,indicating that prevention was at-tributed to apoptotic inhibition by increased levels of GSH-P X ,SOD and GSH.At the highest dose of ascorbic acid and thiamine,the results differed from the low and middle doses.When compared with the only lead exposure group,the combination of high dose VC (1260mg kg À1)and VB 1(90mg kg À1)had no effect on lead toxicity of oxidative damage.We also previously found that this combination caused more severe injury in testis than that in lead-treated group (Wang et al.,2006).Although ascorbic acid was a well-known antioxi-dant,it might possess pro-oxidant property under certain conditions.When the concentration of ascor-bic acid is higher than normal,ascorbic acid level still kept normal in plasma by way of self-metabolism indeed continued to act as antioxidant scavenging free radicals.However,excessive concentration led to accumulation of ascorbic acid,and then an in-creased level of lipid peroxidation (Childs et al.,2001).The pro-oxidant character of ascorbic acid was due to its high reactivity with iron (Halliwell and Gutteridge,1999).Ascorbic acid,as an electron donor of chemical reaction both in intracellular and extracellular states,provided an electron to Fe 3þwhile the concentration of ascorbic acid was exces-sive.The reduction potentials of Fe 3þand ascorbic acid easily allow for the formation of the ascorbateradicals and Fe 2þiron,which generated O 2À,OH Àand H 2O 2.The free radicals increased lipid peroxida-tion,oxidize GSH and subsequently promoted liver cell apoptosis.Additionally,thiamine is a soluble vi-tamin and has been assumed to be generally non-toxic mainly due to the ready excretion of excess thiamine from the body.In many multi-vitamin supplements for human and mice that were commercially available at present,.100mg of thiamine per 1kg of body weight were added for everyday intake (Franca et al.,2001;Zhou et al.,2003).Thus,we thought that VC had no effect on lead toxicity of oxidative damage at higherdose.Fig.3.Factorial analysis for Olive tail length.(A)Joint action between VC and VB 1and (B)joint action between VB 1and VC.568 C.Wang et al.by guest on March 7, 2011 Downloaded fromCONCLUSIONIn 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