Study On The Potential Carcinogenicity Of Irrigative Wastewater In Shijiazhuang City

To alleviate the shortage of water resources, many countries in theworld utilize industrial and domestic wastewater for irrigation. Thedeveloped countries, such as the United States, Japan and Germany, realizedthe significance of wastewater reuse many years ago, regenerating andtreating for agricultural irrigation, groundwater recharge, landscape ecologywater, industrial water, green irrigation and so on. According to differentreused objects, different strict standards were developed by the United States,and the urban wastewater treatment level is basically above secondarytreatment, with the processing rates up to100%. At present, in the developedcountries, wastewater irrigation is mainly used for gardens, pasture, foragecrops, and for certain cash crops, while less for food crops and vegetablesapplications.China has a shortage of water resources; on the other hand, theemissions of China's wastewater are increasing year by year. To alleviate thewater shortage situation, China initiatively utilizes wastewater for irrigationon a large scale. Urban wastewater irrigation, after being collectively treatedby the sewage plants, can save massive water resources, improve waterre-utilization rate, alleviate water pressure, fully take advantage of thenitrogen, phosphorus resources and reduce the usage of fertilizers. However,low treatment rate and low level of sewage treatment exsit in China.Domestic and international studies have confirmed that the sewage containsa variety of xenobioties such as metals (lead, chromium, cadmium) andorganic pollutants (polychlorinated biphenyls, phthalates, polycyclicaromatic hydrocarbons, heterocyclic compounds). These pollutants can notcompletely be removed by conventional sewage treatment. Utilizingwastewater containing metals and organic pollutants in agriculture irrigation, may lead to the accumulation of metals and organic pollutants in the soil, andthere is increasing trend year by year, resulting in the decline of soilproductivity, crop production or even no yield. Also, these chemicalcontaminants with the characteristic of lipid solubility, hardly degradationand easily bioconcentration, can enter into the human body through the foodchain and then pose the potential hazards to human health. Once drinkingwater or food contaminated by metals are ingested by human, the body ofnecessary trace elements will be consumed, resulting in a series of healthproblems, such as immune dysfunction, intrauterine growth retardation, thehigh incidence of nutritional deficiency-related disability and malignanttumor and etc.Shijiazhuang City of Hebei Province is also one of the main wastewaterirrigation areas in China, Mainly including several areas of the countieslocated in the south of the city with a total area of about438.8km2. Tworetrospective studies of the population cause of death in wastewater irrigationareas were done by our department. One study in1980s found that all-causestandardized mortality of sewage irrigation area was623.71/105,significantly higher than557.22/105of the control irrigation area; and cancerstandardized mortality of sewage irrigation area, significantly higher than144.57/105of the control irrigation area, was155.45/105, with an increasingtrend from145.79/105in1977to200.59/105in1982. Another retrospectivestudy was conducted by our department in2008. It found that all-causestandardized mortality of sewage irrigation area was525.78/105,significantly higher than453.14/105of the control irrigation area; cancerstandardized mortality of sewage irrigation area was significantly higher thanthat of the control irrigation area. The results of the studies showed thatwastewater irrigation might be a very important factor responsible for thesignificantly higher cancer mortality of the people lived in sewage irrigationarea than both that in control irrigation area and the national average,suggesting that irrigative wastewater may be potentially carcinogenic. Basedon epidemiological survey results, it is necessary to further research on the potential carcinogenicity of irrigative wastewater in order to standardize themanagement of wastewater irrigation, and to achieve the purposes ofenvironment protection, disease prevention and health promotion.Part one: Analysis of metals and organic pollutants in irrigativewastewate from Shijiazhuang CityObjective:Analysis the main components in irrigative wastewater fromShijiazhuang CityMethods: The concentrations of metals in irrigative wastewater inShijiazhuang City were determination by Inductively Coupled Plasma MassSpectrometry (ICP-MS). Organic pollutants were qualitative analysis by GasChromatography Mass Spectrometry (GC-MS) after water sample wasextracted by solid-phase extraction method.Results: The concentrations of metal elements (Cr, Mn, Ni, Be, As, Cdand Pb) in wastewater were detected by ICP-MS by adding the internalstandard method to correct the interference effect. The recovery rates werebetween96%and102.3%, and relative standard deviations (RSD) were lessthan3%, indicating good accuracy and precision of the method. Theconcentrations of Mn,Ni,Be,As,Cd in water samples were in line with thereuse of urban recycling water-Quality of farmland irrigation water(GB20922-2007). The chromium concentrations of the upstream andmidstream specimens exceeded the national standard by2.3and0.1fold,respectively; the lead concentrations of upstream and downstream specimensexceeded the national standard by0.83and0.55fold, respectively. Thechromium and lead concentrations of the composite sample exceeded thenational standard by0.78and0.43fold.86peaks were detected by GC/MS.In light of the chosen peaks of credibility above70%, a total of30kinds oforganic compounds were detected, including phthalates, heterocycliccompounds, polycyclic aromatic hydrocarbon compounds and anilinecompounds.Conclusions: The concentrations of chromium and lead in irrigativewastewater of Shijiazhuang exceeded the national standard. A total of30 kinds of organic compounds were detected, including many organiccontaminants which are confirmed human carcinogens or suspectedcarcinogens.Part two: Short-term carcinogenic effects of irrigative wastewaterfrom Shijiazhuang CityObjective:Short-term carcinogenic effects of irrigative wastewaterfrom Shijiazhuang City were evaluted by applying a battery of short-termcarcinogenicity bioassays.Methods: The short-term carcinogenicity bioassays of different geneticendpoints were chosed.Salmonella typhimurium reverse mutation assay (Ames): The Amesassay was performed using the standard plate incorporation method and alsoby the pre-incubation procedure. A mixture containing0.1ml wastewatersample of different concentrations and0.1ml test strain was initiallyincubated at37℃for20min in culture tubes (If metabolic activation wasrequired, the S9mixture of0.5ml was added too). Subsequently the mixturewas added to a tube containing2ml of top agar with0.5mM biotin-histidinewas then gently vortexed and poured onto a minimal glucose plate. Aftersolidification of the top agar, the plates were turned over. After incubation at37℃for48h, the numbers of revertant colonies were counted. Four differentconcentrations of wastewater sample were examined:25μl/plate,50μl/plate,75μl/plate,100μl/plate. The samples were tested with and without S9mix.The solvent control, positive control and untreated control were alsoconducted at the same time. Triplicate plates were done for each dose andrepeated twice. A compound was considered a mutagen if there was atwofold or greater increase in the number of revertants compared with thenumber of spontaneous revertants and a dose-related increase in the numberof revertants for one or more strains.Micronucleus test of mice bone marrow polychromatic erythrocytes(PCE-MN):Kunming mice were randomly divided into five groups, eachof which has10, five males and five females. The negative and positive control groups received distilled water and intraperitoneal injection ofcyclophosphamide (40mg/kg), respectively. The other three groups receivedirrigative wastewater ad libitum by drinking water, with the concentrations of25%,50%, and100%wastewater, respectively. Two sampling points werechosen. One was harvested after two consecutive days exposed towastewater; the other was derived afte15consecutive days of subacuteexposure to wastewater. Mice were sacrificed by cervical dislocation method,subsequently sternums were taken. After smeared, all slides were air-dried,fixed in methanol and stained with10%Giemsa stain for10min. All slideswere observed under oil microscopic lens. Micronucleus was counted in ablind manner. A compound can be considered as positive result ifmicronucleus were statistically significant compared with negative controlgroup and a dose-related increase in the number of micronucleus wasobserved.Single cell gel electrophoresis assay (SCGE): Kunming mice wererandomly divided into five groups, each of which has8, four males and fourfemales. The negative and positive control groups received distilled waterand intraperitoneal injection of phenobarbital sodium (140mg/kg) which isknown chemical with DNA primary lesions, respectively. Three groupsreceived irrigative wastewater ad libitum by drinking water, with theconcentrations of25%,50%, and100%wastewater, respectively. Mice weresacrificed by cervical dislocation method and then liver cell suspension withcell density of104~105/ml was prepared.The slides were prepared by the conventional sandwich method ofcomet assay. The coverslips were removed gently and the slides were thenimmersed in cold, freshly prepared lysing solution. Protected from light, theslides were left to stand at4℃for1h and then placed in electrophoresisbuffer at4℃for30min to allow the DNA to unwind before electrophoresis.Electrophoresis was performed for20min at25V and300mA in dark atroom temperature. After electrophoresis, the slides were fixed in methanolfor10min. The slides were then submerged in Milli-Q water for3min, repeated three times. The slides were stained with3.2%of AgNO3for30min,and then developed by chromogenic reagent at4℃for5min. The slideswere decolorization with methanol for5min. For each animal,100cells wereevaluated. The olive tail moments were observed under ordinary microscopeusing comet analysis software CASP.Results:Ames: Without S9metabolic activation system in vitro, the number ofrevertant colonies of TA98induced by different concentrations of irrigativewastewater was more than twice that of the untreated controls, and thedose-response relationship was observed(y=34.833+113.133x, rs=0.976,P=0.000). With S9metabolic activation system in vitro, the number ofrevertant colonies of TA98induced by different concentrations of irrigativewastewater was more than twice that of the untreated controls, and thedose-response relationship was observed (y=42.833+124.133x, rs=0.954,P=0.000). Irrigative wastewater in Shijiazhuang City had mutagenicitywhich contained direct and indirect frameshift-type mutagens.PCE-MN: Micronucleus rates of mice PCE exposed to irrigativewastewater for two consecutive days were significant different among groups(F=171.746, P=0.000). Micronucleus rates of positive control group weresignificantly increased compared with that of negative control group(P<0.01). Yet, micronucleus rates induced by different concentations ofirrigative wastewater were not significantly different compared with thatinduced by negative control (P>0.05). After the mice were exposed toirrigative wastewater for consecutive15days, PCE-MN rates of mice weresignificant difference (F=71.706,P=0.000). PCE-MN rates of mice given25%irrigative wastewater were not significantly increased compared withthat induced by negative control (P>0.05); PCE-MN rates of mice given50%and100%irrigative wastewater were significantly increased (P<0.01), andthere was the dose-response relationship (y=0.003+0.007x, rs=0.814,P=0.000).SCGE: After the mice were exposed to irrigative wastewater for consecutive15days, Olive tail moment values were significantly differentamong groups (=35.815, P=0.000). Olive tail moment values of the micegiven25%irrigative wastewater were no significantly increased comparedwith that of negative control (P>0.05). Olive tail moment values of the micegiven50%and100%irrigative wastewater were significantly increasedcompared with that of negative control (P<0.01), and dose-responserelationship was observed (y=0.298+4.055x,rs=0.905,P=0.000).Conclusions:1The results of Ames showed that direct and indirect frameshift-typemutagens existed in irrigative wastewater from Shijiazhuang City.2The results of micronucleus test showed that substances accumulationof irrigative wastewater might occur in mice and irrigative wastewater couldcause chromosomal damage.3The results of comet assay showed that irrigative wastewater fromShijiazhuang City could cause DNA strand breaks of mouse hepatocytes,resulting in DNA primary lesion.Part three: Enhancement of preneoplastic lesion yield by irrigativewastewater in Shijiazhuang in a rat liver medium-term carcinogenesisbioassayObjective: On the basis of the positive results of the short-termcarcinogenicity test, rat liver medium-term bioassay was applied for furtherresearch on the carcinogenic effects of the irrigative wastewater fromShijiazhuang City.Methods: Healthy male SD rats of6-week-old were randomly dividedinto five groups and maintained in the animal facility of experimental animalcenter of Hebei Medical University Affiliated4th Hospital. After one weekacclimation period, the experiment was started. The two stages of rat livermedium-term carcinogenesis model were chosen. At the commencement, rats(group A) were intraperitoneally injected with0.9%physiological saline.Rats (groups B-E) were intraperitoneally injected with200mg/kg bodyweight of diethylnitrosamine (DEN) which is well-knowed initiator of liver carcinogenicity. After two weeks, irrigative wastewater was given by freedrinking ad libitum at doses of0,25%,50%and100%wastewater in groupsB-E and rats in group A were given distilled water. At the end of week3, allrats were underwent two-thirds partial hepatectomy. Rats were continuouslyexposed to different concentrations of irrigative wastewater after restorationof two days. During the experimental period, body weights, foodconsumption and water intake were recorded weekly. The experiment wasterminated at week8. Rats were decapitated after being weighed and bloodsamples were collected for biochemical analysis. The liver of rats wascarefully separated and weighed. Parts of the liver specimens were directlyfixed in paraformaldehyde, and the other part frozen in liquid nitrogen andstored at-80℃refrigerator. Serum concentrations of alanineaminotransferase, aspartate aminotransferase, alkaline phosphatase andgamma-glutamyl transfer peptide enzyme were determinated according toNanjing jiancheng kit. Liver tissue lesions were observed by HE stained.GST-Pi mRNA expression of rat liver was detected by RT-PCR; GST-Piprotein expression of rat liver was detected by immunohistochemistry andwestern blot.Results:1General observation and changes of organ coefficients: At week8when the experiment was terminated, weights of the rats were significantlydifferent among groups (F=27.925,P=0.000). Weights of the rats in negativecontrol and given25%irrigative wastewater and were no significantlyreduced compared with that of DEN control group (P>0.05). And weights ofthe rats given50%and100%irrigative wastewater were significantlyreduced compared with that of DEN control group (P<0.01). With theincreasing doses of wastewater, weights of the rats were significantlyreduced (y=414.146-53.136x, rs=-0.822, P=0.000). Organ coefficients ofthe liver among groups were significantly different (F=6.961,P=0.000).Liver Organ coefficients of the rats in negative control and given25%irrigative wastewater were no significantly increased compared with that of the DEN control group (P>0.05). Liver organ coefficients of the rats given50%and100%irrigative wastewater were significantly increased comparedwith that of the DEN control group (P<0.05). With the increasing doses ofwastewater, liver organ coefficients of the rats were significantly increased(y=2.935+0.359x, rs=0.459, P=0.004). No significant difference wasobserved in food consumption and water intake (P>0.05).2Liver general observation and pathological morphological alteration:Liver tissue of HE stained tissue sections was observed at high magnificationunder general microscope. The liver of the rats given25%irrigativewastewater showed a small amount of proliferation of oval cells in portalarea. Of the rats given50%irrigative wastewater, organizational structures ofhepatic lobule were destroyed, showing regeneration nodules and small cellfoci; of the rats given100%irrigative wastewater, organizational structuresof hepatic lobule were destroyed seriously, forming fibrous tissues andpseudolobule, and a large number of oval cell proliferation in portal area.3Effect of irrigative wastewater on liver function: AST serumconcentrations of the rats were significantly different among groups(=35.735, P=0.000). Compared to the DEN control group, AST serum levelsof the rats in negative control and given25%irrigative wastewater were nosignificantly increased (P>0.05); AST serum levels of the rats given50%and100%irrigative wastewater were significantly increased (P<0.01) and thedose-response relationship was observed (y=57.620+248.277x, rs=0.928,P=0.000). ALT serum concentrations of the rats were significantly diffentent(=38.684, P=0.000); ALT serum concentrations of the rats in negativecontrol group were no significantly higher than that of the DEN controlgroup (P>0.05). ALT serum concentrations of the rats given differentconcentrations irrigative wastewater were significantly higher than that ofthe DEN control group (P<0.01) and dose-response relationship wasobserved (y=14.540+246.921x, rs=0.960, P=0.000). γ-GT serum levels ofthe rats were significantly different (F=12.987, P=0.000). γ-GT serum levelsof the rats in negative control group and given25%,50%irrigative wastewater were no significantly increased (P>0.05); γ-GT serum levels ofthe rats given100%irrigative wastewater were significantly increased(P<0.01). No significant difference was observed of AKP serum levels of therats (F=1.824, P=0.144).4GST-Pi mRNA expression of rat liver: Rats initiated by DEN, mRNAexpression of GST-Pi in the rat liver tissue were significantly differentamong groups (F=37.904, P=0.000). Compared with that of the DEN-treatedgroup, GST-Pi mRNA expression of the rats in negative control group andgiven25%irrigative wastewater were not significantly upregulated (P>0.05);GST-Pi mRNA expression of the rats given50%and100%irrigativewastewater were significantly upregulated (P<0.01). GST-Pi mRNAexpression of the rat liver were increased in a dose-dependent manner(y=1.029+1.060x, rs=0.950, P=0.000).5GST-Pi expression of rat liver: Results of western blot showed lowGST-Pi expressions of the rat liver were observed in negative control andDEN-treated control. Rats initiated by DEN, GST-Pi expression of the ratliver were significantly different (F=268.176, P=0.000). Compared with thatof the DEN-treated group, GST-Pi protein expression of the rats in negativecontrol group were not significantly upregulated (P>0.05); GST-Piexpression of the rats in different concentration wastewater groups weresignificantly upregulated (P<0.01). With the increasing concentrations ofirrigative wastewater, GST-Pi expression levels were upregulatedsignificantly (y=0.470+0.823x, rs=0.972, P=0.000).GST-Pi protein expression was not observed in the negative controlgroup by immunohistochemical method. Rats initiated by DEN, GST-Piexpressions of the rat liver in DEN control group were low. Rat givendifferent concentrations wastewater afer initiated by DEN, GST-Pi proteinexpression were significantly increased compared with that of the DENcontrol in a dose-response manner and it was highest in100%wastewatergroup.Conclusions: Irrigative wastewater in Shijiazhuang City has the function of tumor promotion. By the effect of irrigative wastewater inShijiazhuang City, significantly weight losses of rats were observed, liverorgan coefficients of the rats were significantly increased and abnormal liverfunctions were observed. GST-Pi mRNA and GST-Pi expression levels of therat liver induced by irrigative wastewater of different concentrations weresignificantly upregulated in the dose-response manner.Part four: Effect of CYP1A1and CYP1B1on rat livermedium-term carcinogenesis bioassay of irrigative wastewaterObjective: To study the effect of CYP1A1and CYP1B1on rat liver inmedium-term carcinogenesis bioassay of irrigative wastewaterMethods: Animal model was same as the third part. CYP1A1andCYP1B1expression at the mRNA and protein level were examined byRT-PCR and western blot.Results:1CYP1A1mRNA and protein expressions of rat liver induced byirrigative wastewater: CYP1A1mRNA expression of the rat liver wassignificantly different (F=73.054, P=0.000). Compared to that of theDEN-treated group, CYP1A1mRNA expressions in the liver tissue of therats in negative control group were not significantly upregulated (P>0.05);CYP1A1mRNA expressions in the liver tissue of the rats given irrigativewastewater of different concentrations were significantly upregulated(P<0.01) in a dose-dependent manner (y=0.346+0.621x, rs=0.941, P=0.000).CYP1A1expressions of the rat liver were significantly different (F=6.788,P=0.007). Compared to that of the DEN-treated group, CYP1A1expressionsin the liver tissue of the rats in negative control group and given25%irrigative wastewater were not significantly upregulated (P>0.05); CYP1A1expressions in the liver tissue of the rats given50%and100%irrigativewastewater were significantly upregulated (P<0.05) and a dose-responserelationship was observed (y=0.557+0.316x, rs=0.799,P=0.001).2CYP1B1mRNA and protein expressions of rat liver induced byirrigative wastewater: CYP1B1mRNA expressions of the rat liver were significant different (F=11.539, P=0.001). Compared to that of theDEN-treated group, CYP1B1mRNA expressions in the liver tissue of therats in negative control were not significantly upregulated (P>0.05);CYP1B1mRNA expressions in the liver tissue of the rats given irrigativewastewater of different concentrations were significantly upregulated(P<0.05) in a dose-dependent manner (y=0.527+0.313x, rs=0.907, P=0.000).CYP1B1expressions of the rat liver were significant different (F=8.247,P=0.003). Compared to that of the DEN-treated group, CYP1B1expressionsin the liver tissue of the rats in negative control group were not significantlydifferent (P>0.05); CYP1B1expressions in the liver tissue of the rats givenirrigative wastewater of different concentrations were significantlyupregulated (P<0.05). CYP1B1expressions were significantly upregulatedwith the increasing doses of irrigative wastewater (y=0.530+0.528x,rs=0.789, P=0.002).Conclusions: Irrigative wastewater in Shijiazhuang City had thefunction of the induction of abnormal expression of cytochrome P4501A1and P4501B1and played the role of tumor promoters, eventually resulting inthe precancerous lesions of the rat liver...