Document 107MpQVXjk2v4OQNj5D08MvjX

FOR DU PONT USE Nanjing Yaoxueyuan Xuebao 5,no.12:114-7 (1985) TRANSLATION This maters may be protected by copyright Law (Qh 1Z. Ud. Code), EFFECT OF LEAD ON GENITAL-CELL CHROMOSOMES IN THE MOUSE Huang Xupei, Xu Juanhua (Department of Hygienics,, Naujing Institute of Medical Science), Wang Xiaoping (Graduate student), Fong Zhiying (Supervising professor), and Qu Weilei (Supervising professor) Received July 7, 1984 Abstract Male mice divided into four groups were naturally fed with waters containing 0, 500, 1000,* and 2000 ppm of lead acetate for a continuous 50-day period. Their average blood-lead contents were subseguently determined to be 12, 42, 71, and 87 iig/dL, respectively. The rates of chromosome aberration and sisterchromatid exchange of., the spermatogonia cells in these mice were examined by the BrdU experimental method. The results obtained indicate that the long-term exposure of a male mouse to. a high dose of lead acetate would very possibly induce permanent damage to the genetic substance of its genital cells. Key words: lead acetate, mouse, spermatogonia, 5-bromodeoxyuridine, chromosome aberration, sister-chromatid exchange. Lead is a common toxic element seen in industrial and environmental pollutants. In recent years, many studies have been conducted on its genetic effects in the human body both at home and abroad. The results of some animal tests have demonstrated that lead can cause stillbirths and/or teratism in Mus musculus, Rattus norvegicus, and Citellus [1,2] . The number of abnormal spermatogonia cells in mice fed food containing 1% lead acetate for 8 weeks was found to be significantly higher than that in mice of a control group [3]. After an extensive epidemiological N 27025 2 survey, Browder et al. reported that the number of premature births and abortions was found to be increased in women who themselves or their husbands were engaged in professions which involve the use of lead; the number of stillbirths was also found to be increased in women who themselves or their husbands Were positively identified to be subject to lead poisoning [4]. ' Although there have been a handful of publications reporting observations of increases in the periphery of the hemelymph micronucleus, chromosome aberration, and sister-chromatid exchange (SCE) rates in humans caused by lead contact and lead poisoning [5,6], as well as one article describing an animal experiment, in which the rate of myelochromosome aberration was found to be increased in the mouse, rat, goat and monkey exposed to excess lead [7], there has been no study reported on the effect of lead on genital cell chromo somes . It is common knowledge that once the genetic substance in genital cells is damaged, the effect will be passed on for generations. In this sense, the examination of the genetic toxicology of lead through the determination of its effect on genital cell chromosomes appears to be very important. In our present study, mice divided into several groups were fed lead- containing water for a certain period of time, their blood-lead contents were subsequently measured, and the variations in the spermatogonia cell1s SCE and chromosome aberration rates were determined. Materials and method I... Materials Healthy and mature male mice with body weights of 20 to 25 g were used as the experimental results. 15 mice were randomly divided into 4 groups, with the control group consisting of DUP040006245 3 6 mice. Testing groups I, II, and III were given drinking water (deionized) containing 500, 1000, and 2000 ppm of lead acetate (corresponding to 275, 546, and 1092 ppm of lead) respectively, while the control group was only given plain deionized water. The mice were allowed to freely drink the water for a continuous period of 50 days. At the 48th day, all mice were given 0.6 mL of BrdU (Fluka) solution adsorbed onto activated carbon once through abdominal injection. The preparation of the carbonadsorbed BrdU solution was carried out by Kanda's method [8] with a slight modification. Twenty g of activated carbon were first ground in an agate mortar into a fine powder, then suspended in 500 mL of distilled water. After testing for 5 min, the upper part of the suspension was transferred and it was centrifuged at 2000 rev/min for 8 min to separate out the fine activated carbon powder. This fine powder was then washed with 2.5% NaOH, distilled water, and 2.5% HC1 in sequence for five times and finally washed with distilled water again to neutralize. The suspension was again centrifuged and the collected precipitate was dried and disinfected at 180C for 30 min. In a separate operation, 10 mg/mb of BrdU aqueous solution were prepared, filtered, and disinfected. The BrdU aqueous solution was added with the disinfected activated-carbon powder at a concentration of 50 mg/mL. The solution was fully stirred at room temperature for 1 hour prior to use. Twenty-six hours after the injection of BrdU, 3 mice taken from the control group which was fed 0 ppm of lead were administered mitomycin C (0.4 yg/g body weight) once through abdominal injection and they were used as a positive reference. The remaining 3 mice in the control group were used as a negative reference. Fifty-four hours after the injection of BrdU, all of the mice in the test and control groups were given colchicine through abdominal injection (4 mg/kg body weight) and they were sacrificed 4 hours later. DUP040006246 4 II. Det.eqiilna.tion of blood-lead content The blood samples were collected through the excision of the eyeballs while killing the mice. .After the anticoagulant treat ment with heparin and homogenization by shaking, the lead contents of the blood samples were determined with a Hitachi 180-80 atomic absorption spectrophotometer using a graphite furnace* III. P^ggaration^f^chromosome^sgecimen The chromosome specimens were prepared by a modified Evan and Meredith method. After removing the testis, the. white membrane was cut off and the convoluted tubules were hypoosmoti- cally treated with 1% sodium citrate at room temperature for 30 min, A mixed solvent of acetic acid and methanol (1:3) was used to fix this sample and a 60% acetic acid solution was used for softening. The tubules were then broken by blowing to form a cell suspension. After centrifuging the suspension at 1000 rev/min, the specimen was prepared by the regular gas-dry method. A part of the chromosome specimens were used for the sister-chromatid difference (SCD) treatment by the UpG chromatic method, IV. Chromosome_observation and rate counting The SCE frequency of the spermatogonia cell was counted by the regular method. For each mouse, 25 chromatically clear metakinesis phases were counted. The exchanges at the terminal of the chromosome and the centromere were counted once and the exchange at the middle of the chromosome was counted twice. When dealing with the chromosome aberration of the spermatogonia cell, 100 metaphases were counted for each mouse; the aberrations involve chromatid and chromosome breakage, space, depletion and fragmentation. DUP040006247 5 Results The sister-chromatid difference is clearly Seen in the meta chromosome picture of the mouse spermatogonia cell after the UpG treatment conducted 54 hours after the abdominal injection of BrdU adsorbed onto activated carbon (Figure 1). All chromosomes had a relatively light chromatid combining with BrdU. Figure 2 shows the phenomenon of chromosome aggregation between multiple cells. This phenomenon was seen in both the negative control group end test groups and therefore it may not necessarily reflect the change in chromosome number. In the literature,, the phenomenon has been explained as a result of the mutual aggregation between the meta chromosomes of adjacent synchronous spermatogonia cells and it is considered to be a normal phenomenon associated with the periodic and synchronous splitting and differentiation process of the male genital cells [9]. For the above reason, the statistics of such chromosome aggregations have not been studied in our current experiment. I Figure 1 Figure 2 The observation results for the SCE rates of the mouse spermatogonia cells are collected in Table I. The average SCE rate of each cell in the negative control group was 1.70 + 0.13; DUP040006248 7 difference in the chromosome aberration rate was seen.only in the 2000~ppm group and mitomycin C group (p < 0.05), but not in the other groups (p > 0.05). Table II. Observation results for chromosomal aberrations of spermato gonia cells in mice after drinking lead-containing water (for 50 days). Chromo- p. a o p o * 00 d U 0 Pft o *d 0 y 0 ^3 .ft H d as 0 ft a. e H o 0 u ft, .a 4-1 a* 3 c--$ ft w S3 P -g $00 0 h +J 60 1 0 1-4 <0 d <y d u o w <++ 0 o 0 69 >H o a < rO O w Chromated ta a H <u p iH t <u d ft- U 8 83 a m ft ss 0 O d 60 0 a: 0 0 * 0 0 0 P* 00 .ftso'me '<O0=!OswH H P w0 A! 0 0H 0H0 P 0 04 0 0 0 w O< PaOif* rt /-n 4J fc* 0 -w U0 0 4J Negative I It MMC 0 soo 1000 2000 0 4 4 4 4 4 12 400 -- 2 -- - -- 0.5 42 400 1 8 -- -- -- 1,0 n 400 s 4 -- -- - -* 1.5 87 400 1 8 -- 1 1 -- 2.2** 400 2 5 -- 2 1 -- 2.5'* * One mouse was added to each group and it was treated in the same manner as the previous mice. ** Statistical analysis exhibited a P value of less than 0.05. Discussion There have been some studies reported on the effect of lead bn the chromosome aberration and SCE of some mammal cells (myelogram and-hemolymph). Its effect on the chromosomes and SCE of genital cells, however, has not been well understood. The genital cell is the direct hereditary material and it is therefore an important research object in genetic toxicology. The success of the BrdU internal test method made the in vivo determination of SCE in mouse spermatogonia cells useful for an effective and sensitive detection of induced mutation by toxic substances. At the present time/ SCE is considered to reflect the breakage and restoration processes of DNA chains. The SCE DUP040006250 6 those in the 500-ppm group and 1000-ppm group were 1.68 + 0.18 and 2.11 + 0.14, respectively. Compared to the negative control group, the 500-ppm and 1000-ppm groups did not exhibit signif icantly different results (p > 0.05). The average SCE rate of each cell in the 2000-ppm group was 2.84 + 0.29; that in the mitomycin C group was 5.04 + 0.69. Compared with the negative control group, these two groups had significantly different results (p < 0.01) . Table I. Comparison in SCE rates of spermatogonia cells in mice after drinking lead-containing water (for 50 days). Group O fl) wo cad) ui0d) Q) o S ppui Q cd ' u <al> /-<v* 0)a> ri o * 9 JS O.'w' T3 <D <P r-i 4J 00 1 d u *,oo0 e0) u -ijo) t>0 > H O 3L <$ M V V" <W rH a> n ,a0) .J25 H ao) MoH >u0CO) o , .SCE frequency ucd p1 Jtt>ol0 ao) W. ->0<) WCWJ I^X t test Negative I n nt MMC 0 Soo loco 2C00 0 3 . 13 S <4 3n 3 37 3 76 7S 75 76 81 129 1 *7C 0.13 126 1.694 0.18 148 1.99 0.24 216 2.84 5.29 408 6,04 0.69 >.C. C5 >0,05 <0.0! <0,0! The observation results for the chromosome aberrations of the mouse spermatogonia cells are collected in Table II. In the negative control group, only 2 cells with a chromatid space were counted, with the aberration rate being 0.5%. In the 500, 1000, 2000 ppm and mitomycin C groups, the Observed aberration rates were 1.0%, 1.5%, 2.3%, and 2.5%, respectively. The chromosome aberrations counted involved spacing, breakage, and depletion, with the space type of aberrations as the most frequently encountered. The result of the statistical analysis indicated that in comparison with the negative control group, a significant DUP040006249 8 determination of mammal cells often serves as an effective way of detecting mutagens and carcinogens [10]. In our present experiment, the BrdU internal test was carried out by Kanda's adsorption method, using a reduced amount of activated carbon (from 100 mg/mL to 50 mg/mL). The obtained results were satisfactory. The SCE rate for the mice in the negative control group was found to be 1.70/cell, close to the spontaneous SCE rate of mouse spermatogonia cells (1.3-2.18/cell) reported abroad [8]. The cell's SCE rate, however, was found to be significantly increased for mice in the positive control group to which mitomycin C was injected. Since the concentration of BrdU used in our experiment was very low (6 mg/mouse) and more over its release from the activated carbon surfaces was quite slow, the direct effect of BrdU itself on SCE can be essentially neglected. Our experimental results have proven that the condi tions selected here cannot only save BrdU, but also make the method simpler and more reliable. As indicated by our experimental results, when a mouse was fed water containing lead acetate for a long period of time, its blood-lead content increased with the amount of lead introduced. For the mice that drank water containing either 500 ppm or 1000 ppm of lead acetate for 50 days, the chromosome aberration and SCE rates of their spermatogonia cells were not found to be significantly increased. A significant increase in the chromosome aberration and SCE rates was found forthe mice that drank water containing 2000 ppm of lead acetate. The observed aberration was, however, the chromosome space type in most cases, indicating minor DNA damage. Although the increase in the SCE rate of the mouse s p erma to go nia cells induced by a high content of lead was quite significant (p < 0.01), the SCE number counted for each cell did not reach twice the SCE number in a spermatogonia cell DUP040006251 9 found for the negative-control group mice. According to the criterion proposed by Taft et al., those agents which induce SCE at less than twice the SCE number originally found for a control cell should be considered to be Weak SCE inducing agents [10]. Our experimental results therefore suggest that the long-term exposure of a male mouse to lead acetate at a relatively high dose could cause damage to its genital cell chromosomes, and the induced mutation of mouse spermatogonia cells by lead appeared to be a relatively weak one. To answer the question as to whether the observed mutation is related to sperm irregularity, teratism, and stillbirths caused by lead over exposure and lead poisoning, further investigations are needed. So far, the mechanism by which lead induces mutation is still unclear. Some researchers think that because lead is a heavy metal element, it can undergo combination reactions with some biological materials present in the body. Nucleic acids, for example, contain purine bases and phosphoric acid which possess high reactivities with metals. Among purine bases, guanine and adenine have N, OH, or NH2 functional groups which can readily react with many metals. Once a metal element enters a biological entity, it combines with the basic groups of the nucleic acid. Consequently, a change in the stereoscopic structure of the nucleic acid and a mismatching of the bases could be induced. These alternations of the nucleic acid may influence the genetic property of the cell and cause aberrations and cancer in the biological entity. .At the present time, a tendency to replace old morphological viewpoints with modern molecular biological viewpoints has been formed in evaluating the toxicities .of industrial and environ mental pollutants. The evaluation of toxicity has been carried out based on genetic considerations. The results of our present DUP040006252 xo experiment provide useful reference data for the evaluation of the genetic toxicity of lead. Acknowledgement The authors wish to thank Prof, Zhu Qiding of the Research Group of Embryology, Prof. Huang Yuquan of the Biology Department of Jiangshu Teacher's College, and Prof. Yang Yongnian of our department for their helpful discussions. The chromosome pictures were taken with the assistance of Mr. Sha Jiahau. References I. Gerber GJ3. et al. Toxicity. mutagenicity and teratogenicity o; lead. Wut Res 1980, 76*115. [2] Zhang Guohe, "Toxic effect of lead on embryo," .Guowai Yixue Weishengxue Fenche, 1984, 3 s133. [3] Qu Weilei, "Long-term effect of lead acetate in food on mouse spermato gonia cells, body weight, and life," Guowai Yixue Weishengxue Fenche, 1979, 6(2):119. 4. Ann A Browder, et al, Tbe problem of lead poisoning. Medicine 1973, 52 (2 ) *121. [5] Liu Mingzi et al., "Method and application of sister-chromatid exchange determination," Zhonghua Yufang Yixue Zazhi, 1983, 17.(1):43. [6] Qin Shizhen, "Sister-chromatid exchange--international discussions on the subject," Guowai Yixue Yichuanxue Fenche, 1984, 8:116. [7] Huang Xupei, "Effect of lead on chromosome," Nanjing Yixueyuan Xuebao (Wenxian Zongshu Zhuankan), 1984:30. 5. Raida N, Asimple teebique for in rito obltrration of SCE in mouse ascstestumor and spermatogonia cells. Exp Cell Res 1979, 118 * 451, [9] Yang Yuzhou, "Study on the effect of cottonseed phenol on ehromsome aberrations and sister-chromatid exchange (SCE) rates of mouse spermato gonia cells in vivo," Jiepou Xuebao, 1982, 13(2):217. 10. Latt SA, et al. Sister-chromatid exchanges A report of the gene-tox program. Mat Res 1981, 87 * 17. TECHNICAL LIBRARY NETWORK RALPH MCELROY TRANSLATION COMPANY JANUARY 26, 1989 DUP040006253 av LI 4 Tt ijfipMPUES WITH COPYRirwT i L. c h emk al abs t SI^ r ***** ***** s3y evn^XAd^ H-< A. C .f 9_ _ ***&** * w. 42,71 matpg/dL. fflBrdu &<&m $&> **. mwms i-i**!f, ***, %&&-&&& %& *#r sf^Ms. i%m 8 3 o Browder .&$* iifw, w*mm j r * **> cscE) mmnmw0-mmc5^> m#, m& 20-25go ms^mmMMim., %&m m%6H, u i, i *#500, 1000, 2000ppm <*R*fc27$, 546, 1092ppm) fi&jjfcM;* > j 7X H &*&, m <#503c. fig BrdU CFluka ff) 0.6mlo &uMtott#?few Mmimm8 x &2os *&*, 5ia#j5fa*, aus#* a#60omi3R*t il'5^1. IE 9U** H20008/*-fc 8$j,ja i S)i 2.5% NaoH, ^^;Kfn2.5%HClJ? Sfe 5 &, $ mMmmmmnsim&m, <k '-:spfl mn%'bEfts -asm * MW>um, flmft MftTfeftm u m ft % m ,o^^^i8ot3Tmo*^o a#jB ^10mg/ml BrdU m, &.*, S #3*WJgtt#WSlS0mg/ml80 & jteX 99 1 ;hW. BrtUffltf26*1*, M ppmXfM m*p9ut13 ***&aft-ftjw&a:i4 M*c-(o.4m/g*> #*&** BrdUitfrM54*Bf, a**ife **# ^ 27025.01 ilii'M (4mg/kg{jt>, 4 /b8'\J aswaifeft## m i >,h 2 mmm& JflLiB;185E ^aan^urffli^JiscftjfiL, mm, j b mm iso-80 mmf-mm>%M mmfk, M&m* H, '$& #**(* Evan Rfn Meredith K *#*, mfit 4^ruu%#if#5ft#^ss'r i #30^ #s^ai * 3 60%frZ>mmS, K5fc JT $ &JB looo^/rfl-ftMj^fr, 3feft##*S?3K KopolfefeifeafittftSs^ c s c p) tm, as, as#& mut^mmsmmscEs*, *~s& fA*S^Afc'SEJfeTHS["ac, mm,,ft# mm, m ' ft it teupOAajgTO##3ftftlMjKSIIJte6 m w-^#^Brduife ft m 1 ttffl^*(03c)jB^*JBjBteCEttclfc a *$ I I nt MMG ma ES <ppm> 0 500 1000 2000 9 tftsca%& <p,) 3 3 3 3 3 <ug/dL> 12 42 n 37 .a .m&amsc 76 7-5 75 76 31 sea m && 4F1<3S5CES/) .* 129 1.70 0.13 126 1. G8 0.13 148 1.99 0.24 216 2.84 0.39 40$ 5.04 0,69 >0.05 >0.05 <0,01 <0.01 s c e mm 0,13, 500ppm M 'f' mmr m SCE$.% 1,680.18, 1 000ppmM't'i$SCE 3^2.11 0.14. SOOppmf&lOOOppmMi? $ 2 (5o^) 2000ppma^/haiiaSCE3pitffl H^*2,,84 0.29, ajRffl45*88J|i&SCE$ 5.04 + 0.69, <P<0.01) . ' ffl ^ Hit I I III MMC n *afc.** ##R (PP) <K) 04 509 4 1000 4 2000 4 04 * (ug/bD 12 42 71 87 a$ &JS 400 400 400 400 400 xsi must --2 i0 24 16 25 *ffl?K-K4'R, itaraffl. '&gstf-^##?p<0..05. $& JF ws s _ .- - -- - ---- -- ----- -- 1 1-- - 2 1-- 2t* (X) 0.6 1.0 1.6 2.3** 2.6** UI2K, at3e^0.5%, B0500, 1000, 2000ppmS-&$&&** C 1.5%, 2,3%f[|2.5% 0 &$*, *r> fcSb WK#**J&o ^ ^#^20ooppmiifnt^^c m %$ Cp<o,o5), ttsesatta# (P>o.o5) . $k, <ioomg$M somg/mi), aaraitwm.* #&&* TO*tM/J\ RWs c e Hi . 7o/ffl}!S, EHJSiBjflscE g j.. 3 --2.1.8/ai) mc & mshWtt?a&ttmfsi,mMscE%:m *. &in BrdU^g^^(6mg/ 1 K/hi.), ms.w.miEmmi.m.mtwi, m*Mwmrdv*MmcE&iMm& r. mum, n *& <MfBUSU #s *8SB) 3ft`&##S52lSCE^l!fB,-iit a. m*m &/J> *flpaBscEttwjarHfrit*ai^riF amikfcscE&fcT a^DMAftia ? ^^Hftafi9amci<o,aieaaa fflTKftodaRtt^tt^KISBrdW# ft SMS***. 4fr*Rfctfcfc# % %t 1p500fniOOOppmSll^ sjfimwMWL& M'h Kaxaiaaft -**** ft2000ppmMit m M^&m. KDNASft. ffaaan&ffJKftUBscE <p <o .o d ,a JfcSCE.Wfc, & M##scEfc*i&&*j- MM ^^s c e |S:M#^^s c e ^PH. ^JSjaoa, j m, : $jsi&fii; | mm, :! #. ii. < mfamfc S I =f% &m& i " **Hr; s, a ***; *1E s &#, : gla ` M&U& mmM. Mmum <*X R; ft M1 a* * ^ i | ab ; I 3 aar 0. I UP0400062 mitf m m, mt? *Riaraamntrgs!!m% tfrmMHa mm, * m w& $ WW$#^RJ&`*3&JRRkN, Oh -Rn h * mm, mst, &m-s.mxmm, mm *#& ? ifdnmz&m m ?mmm -mm&, m mMWT0%mm, < *x # *mmmm # w& *& n * , tc*. # ^ jf*^aiR'jgsajaa, $&. > 117 #* * 1. Gerber GB, et al. Toxicity, mutagenicity and teratogenicity of lead. Mnt Res 1980, 76 * 115. 2. 05*:. m K &&&.& m 1984 3 I 133. 3. a*a. tf.iais^4'S'.,#iiiaia]B ttUZMftM, ##:&& 5* 3 ms, 6 (2;). 119. 4. Ana A Browder, et al, The problem of lead poisoning. Medicine 1973, 52 (2) 121. s. *, %.<&&&%.& sawaj:*assa. *J*4pBfg#3SSf,$ 1983, 17 (1) < 43. S*l>Ka#*-* 1984, 8 lift. 7. " M. *!&#&#. K#RJR<3:tt ISSJE^fl) 1984 *30. 8. Kanda N. Asimple techique for in vivo obser vation of SCE i mouse ascitestnmor and spermatogonia cells. Exp Cell Res 1979, 118 431. ft#=3& (SCE) *$&$. M# 1982, 13 ( 2 ) > 217. 10, Latt SA, et al, Sister-chromatid exchanges < A report of the gene-tox program. Mnt Res 19.811 87 17. <19.84#7^7Hft) . m$ m xiim*a m, 63^, m.= p w m ie?m . wmm&j e mm tttm iws& mmm, pim&im&m#0m & (Human Pathoigy 1984, 15 (7) * 857--659, **** =)