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i DISCUSSION Inquirer: Michael Kay, University of Missouri Research Reactor, Columbia, MO Q. How is coal volatilization process different from nuclear weapons de tonation where refractories are on larger particles? A. Clearly, many elements display an increased concentration with de creasing particle size of coal fly ash. The size dependence appears to be due to a variety of factors including size distribution of min eral grains in the coal and the volatility, reactivity and stability of chemical or elemental forms that exist in coal. It is well estab lished that those elements (or their oxides) which are volatilized at coal combustion temps are preferentially concentrated on fine fly ash particles, i.e. concentration of condensed volatilized forms is a sur face related phenomenon. Decomposition reactions, e.g. carbonates or sulfates, may give rise to fine particle formation of relatively re fractory elements. Such fine particles may agglomerate with larger particles, again giving rise to a surface related concentration en hancement. A similar argument pertains to mineral grains which may also give rise to fine particles of relatively pure materials. Chem ical reactions may also give rise to volatile species from refractory compounds. For example, it has been suggested that silicon may be volatilized by reaction of silica with carbon resulting in formation of SiO. Subsequent nucleation processes may give ris~ to submicron agglomerates which deposit on surfaces of larger particles. rV) - .o .ucJ > '.X*ml TM" h f row iffht t a YT.-tlp 17. T7.S. CoSnT 298 R ev ersal of Im m unologic an d Toxicologic E f fe c ts o f a S in g le E x p o s u r e o f 2, 3, 7, 8 -T etrac h lo ro d ib e n z o -p -d io x in in M ice O ft ?(!$ R. P. Sharma Utah State University Logan, Utah s-\ i , ( O -A . R. J. Kociba and P. J. Gehring The Dow Chemical Company Midland, Michigan HA-ftu ABSTRACT A single dose of 10 ug/kg of 2,3,7,8-tetrachlorodibenzop-dioxin (tCDD) in CD-I mice produced signs of toxicity includ ing microscopically detectable degenerative changes and in creased weights in the liver, lymphoid depletion and decreased weights in thymus and alterations of various hematological pa rameters (decreased red blood cell count, lymphopenia and neu trophilia). This treatment also increased spontaneous trans formation of lymphocytes in cell cultures prepared from spleens of treated mice. Transformation of these lymphocytes induced by mitogenic agents was reduced. These effects on lymphocyte transformation, apparent at 2 weeks post-administration, were not discernible at wk 4. By 8 wk post-administration, the wts of liver were approaching normal and the degenerative lesions observed microscopically were less severe; although thymic wts were still depressed, no lesions were microscopically discern ible in this organ. At this time RBC counts were normal while mild lymphopenia and neutrophilia persisted. Transformation of lymphocytes, either spontaneous or mitogen induced, showed no deviation from that of control. These results indicate that in mice the toxicity of TCDD, including untoward immunologic ef fects, is reversible in a relatively short time. CO co o o INTRODUCTION 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic contami nant formed in the manufacture of trichlorophenol and in a number of other related compounds (4,12,19). It is one of the most toxic chemicals known (other than biotoxins). Studies on various toxic effects of TCDD have in cluded teratologic (3), general toxicologic (6), pathologic (5) and other clinical effects (20). It is a potent inducer of hepatic microsomal en zymes (10) and causes induction of -aminolevulinic acid synthetase (9). A 13-wk oral feeding study in rats with different levels of TCDD and re lated toxicologic effects has been reported (7). In addition, there have been implications that TCDD may adversely af fect the immunological system. This chemical was shown to be particularly toxic to lymphoid organs, e.g. thymus (2). Gupta et al. (5) found that fCUD was toxic to the thymus of rats, mice and guinea pigs, despite some selectivity of other organs in these species. Vos et j 1 . (17) reported the immunosuppressive effects of TCDD in rats and mice; subsequently Vos and Moore (16) suggested that the effects were primarily on cellular im mune functions. Thigpen et al. (15) showed a decreased h r 4, "insitivity to bacterial but not to viral infections. In our recent s ?s (reported 299 ^.separately) we have found a decreased responsiveness in mice and rabbits exposed to TCDD when their immune function is challenged with an antigen. Using a splenic cell culture system, however, an increased UNA synthesis in lymphocytes from animals exposed to TCDD was observed. The objective of this study was to determine if the toxicologic ef fects, particularly the immunologic ones, are reversible with time. This report describes the time related effects in mice of a single exposure to TCDD for periods up to 8 wk. MATERIALS AND METHODS Male, CD-I mice were obtained from Charles River (Wilmington, MA) and were acclimated for 1 wk. They were housed 2 per cage, provided free ac cess to feed and water and kept in an environmentally controlled room with a 12 hr light/dark cycle. At the start of the experiment, the animals weighed approximately 28 g each. The mice were divided into 2 groups; half were dosed with a solution of TCDD in corn oil (containing 5% acetone) at the rate of 10 ng TCDD/kg and the other half were dosed with an equal volume of vehicle alone. Groups of 3-4 animals were sacrificed by decapitation at 2, 4 and 8 wk after treatment and blood was collected for total and differential cell counts and also to obtain serum. The liver, kidney, spleen and thymus were obtained and weighed. Spleen was used for lymphocyte cultures; other organs were placed in formalin fixative, processed routinely and examined for microscopic alterations. Splenic cell cultures were conducted as described elsewhere (13). Sus pension of cells obtained from these organs were cultured in triplicate with or without the presence of either phytohemagglutinin (PHA) or pokeweed mitogen (PWM) in micro-culture plates. After 48 hr of incubation in humidified air containing 5% C02, cultures were pulse labeled with 0.5 itCi of 3H-thymidine and incubation continued for additional 16 hr. The cells were harvested, washed and 3H activity counted in a liquid scintillation spectrometer. The counts were converted to disintegrations/min using a standard quench curve. After averaging the replicates for each culture, stimulation indices with both mitogens (dpm with a mitogen/dpm with no mi togen) were obtained and the animals in each treatment group averaged. Serum immunoglobulins were estimated by electro-immunoassay using carbamylated rabbit anti-mouse immunoglobulin in an agarose gel system mod ified from Bjerrum ct al. (1) by a procedure described previously (13). The results are expressed as mean of observations with either stan dard deviation or standard error of the mean. Values in control and treat ment groups were compared by t-test (14), using a prespecified p=.05 value. RESULTS None of the mice died due to treatment or showed any clinical effects of TCDD toxicity. Growth of treated animals was comparable to that of con trols. Body wts, organ wts and organ/body wt ratios for selected organs are listed in Table I. A reduction in thymus/body ratio was observed in the group sacrificed 8 wk after TCDD exposure. The only other organ show ing a change was liver; its absolute wt and the liver/body wt ratio in creased consistently at all intervals, albeit a significant difference oc curred only 4 wk after treatment. The increase in liver wt was nearly 18 at the 2 wk interval, rising to 26% 4 wk after exposure, but decreasing to only 11% by 8 wk after TCDD treatment. Upon microscopic examination treatment related changes were observed in liver and thymus. The lesions were similar to those described else where (13) and the incidence at different time intervals is summarized in Table II. Liver lesions included degeneration or necrotic changes at 2 and 4 w 1' ,ffer treatment whereas fibrosis with increased leukocytic infil-  -- r-- ?! O vr UD O ?! ?l O PO LO LO ? ! +1 o* *3* O+ ,I po +oO ,1 LO LO OO Oc+o,I +O1. LO UD OO o+O1, LO+DI lO LO OO 4-> JC C7> --- CO PU P i LO i-- PO O PO LO PU ^r PO LO <u >> CO ,O-- o, d , 41 + 1 CO d, d, o+ O0 d , o, +1 + 1 P^ P^ "O e n LO LO LO O O LU OO<u")oc O OL)OUJ --I CO Seu O> u -J c O 4--* e n 03 LD LO LT) UD en o PU PO oPO e n ou d , o, o, d , o 41 LT e n C\J oPO U ) LO LO u o C CO PU r-- o, d , oLO 0 0 PU O-zJ a><: 03 OC r" PU PU PU PU PU o--o hLzU- <-- X >- Sr-- a x: h- T3 Ci LO LoD <ox eon o r-- LoO O O o o o, d , o, d +1 +1 4 1 + 1 + 1 \ PO LO LO PO 4- L0 03 4-> ^ ce Q LUU_ C <t3 CO cen <D a> o n PU dd PU PO do PU PU dd L0 4-J -*-> 4-> r -- 03 <O x : CL e n UO LoO o PoU LoO LoO PoU C C LU hc-e h<- LU O O--X LO--U a rs c X3 en 1- en d, d, +1 +1 eon d, d, +1 + 1 eon PU d, o+ oo4,1 ai L0 r-- od od od s_ a; o CD >*--* uoo V O J Z CL O oc LJ o> PoO PoO oo * PU O PoO 4o-> 4-3 *3 LO < f o,h- 00 O O -i-1 o o oX O T3 O d, d, d, d, d, a; r-- + 1 4 +1 +1 4-> \ r> . LT) CO 0 3 r-- L. -- ZLU < LOO l rs r-- do PU oo d d C 4-> 4--> Co h CL U 03 O<Ooc -C 1-- ai oo OoU PU O d , o, d , . d , +! +1 o PoU d, . D X ai 03 > r- 4-J U OZ LoO LoO < oo o ro>* od 0o0 LoO od CL =J O S- 03 OLO. 03 >- 0 3 l_ OOco *>o O ai r--<-'>, ^ . m uO 41+ 1 a.m UO +1 + UD O i + O PU i- ai CL L- CO -- J PO PO PO PO LO oO 0 - a O XI oL_ -o ai 4-J JD O L. JD J J X I "O a> O a +-> L_ E or-- t - C <T3 e n u 4- C UJ 03 oOL- 4- Co LJ hUaL-.i - <Co_> rQLo_J -- 4- Co LJ 0C3J hL-. 1<t4--aaE4l-->;>- t44--CdE00S--*33)>.C0sO3.3 PU KT 00 03 Lo*- CD L/0 CL LO *ETJ aC> > OJ 4O'c-O +1 PO v4-- C >i 2aTJi3 oc JD Ca> LO 301 tratldn was apparent at 8 wk. As seen in Table II, a progressive increase '`W n the incidence of liver effects was observed when tissues were evaluated in a blind random fashion. The thymus effects included regression of cor tical area, depletion of thymocytes and occasional pyknotic foci, but these were seen only in samples obtained at 4 wk after TCDD treatment. A gradual development of thymic changes that took nearly 4 wk, with a rever sal of discernible effects by 8 wk, was evident from the results in Table II. No changes in kidney were observed. TABLE II. INCIDENCE OF LESIONS IN LIVER AND THYMUS OF MALE MICE TREATED WITH A SINGLE DOSE (10 ug/kg) OF TCDD AND SACRIFICED AFTER DIFFERENT INTERVALS3 Duration After Exposure (Weeks) Control Liverb Thymus Treated Liverc Thymus^ 2 0/3 1/3 3/4 0/4 4 2/3 1/3 4/4 2/4 8 2/3 0/3 3/3 0/3 aSections were examined microscopically in blind random fashion. Kidney showed no remarkable change in any group. Numbers indicate incidence of lesions/total number of animals examined in that group. ^Lesions in control liver included vacuolization or slight degeneration of hepatic cells. cLiver in these groups generally showed slight to extensive diffuse degen eration or necrosis. ^Cortical atrophy with depletion of thymocytes seen in thymus of animals sacrificed 4 weeks after treatment. Hematological examination (Table III) revealed a significant reduc tion in the total number of erythrocytes at the 4 wk interval. This change was not noted at 8 wk post-exposure. Total leukocyte counts showed no change at any of the sampling periods. A consistent increase in the number of segmented neutrophils and a corresponding decrease in the number of lymphocytes was' observed at all Intervals. Splenic lymphocyte cultures at different time intervals showed that at 2 and 4 wk after TCDD treatment there was a marked increase in DNA syn thesis in unstimulated cultures. Although the extent of average increase was similar at these 2 periods, there was greater variation at 4 wk, mak ing the difference statistically non-significant (Table IV). At 8 wk, up take of tritium was similar for both control and treated groups. When these cultures were supplemented with either PHA or PWM, the resultant mi togen-induced stimulation was decreased considerably at 2 and 4 wk after treatment with TCDD, but control and treated groups were similar in this respect at 8 wk post-treatment. Estimation of serum-immunoglobulins indi cated that a single exposure of 10 ug/kg TCDD did not produce any marked change in this parameter at either period of sample collection (Table V). DISCUSSION Results indicate that TCDD in a single dose of 10 ug/kg is toxic to fmice and the toxicity is evident by decreased thymus wt, increased liver wt, alterations in hematological parameters and histopathological lesions, o primarily in liver. In general these toxic effects seem to be reversible ^ a n d the reversal may be evident as early as 8 wk after exposure to TCDD. T OO+C1rM+--1 C+M1.*t+----1', rC+--M), TABLE II). SELECTED HEMATOLOGICAL PARAMETERS IN TCDD EXPOSED MICE Mean + SE of 3-4 animals per group ^Stimulation index = dpm in the presence of mitogen divided by dpm without mitogen Significant difference {p<.05) from respective control group CM.CO, CM,.-- , c+m1c+m1 +--1c+vi1 c+o1c+o1 Ut_i> -4-C> r<3>-iMn- CrO-in Ol m O O i-- CM O a z z o Z2 a. J-DX<ou Ci+On|Co+O*1 i--+ 1i+n1 r-*O cm+n1om+o,1 CM>-- =rr*. co c 1- v> r- CO CD CM o -- +aji 3- CO CM O C>J XoT CM CM 4 r-. co O- CO CO tC--O fC--M .. k i-- CO ,r-- O co ir--n 'o'ow- r * rO CM CM, (+n '1c+m 1 O+ C+O CO fM CO in mp+m. >r+--r o 1l/-)<oo0 ozoUJ hoo> H-aX--+c3sn c 4or--> r-- CM CO t-- r-- <d oocn incm tn 0,0, CM,CM, O,o. 4e-><Xo_ to e+nr+ ro--l>Oin I+DC+1 O CM <-- m ri+-n1n0+0I i-- zo tCaoD-nCm-M a i cm 03 cm ooi oCaM-m1i---- rM n c4ot (M. CM lr+--OIl(M+OJ Nr+--1r+-1 >C+--M|tmo| cn <-- I0D1 LOl H<_>VUJ) aoX. oQ >-*j<2o! a-- UI ZU) -o1 --1Z to cacn mm 4if----t cn co CO CD cn cm to cn mUOOn'CMT (U4o3 <_) 31 MO.-43O3C OQ-r4rs3* CM Cl Cci+Mnm1Vcc+mn1 rc-nccanni Cm CM CMLD ,0CM, <c+0n-1ci+nn \O4>-f^C+Ol rin co ocnaCiMnj CCMMCOM +-- 1co+n1 rO+-.1'c+--si. 0,0 . Ol/-)--to >- t. co uj g ii---- mm UJ 1Ll-. CM IiDn CcOo cu ou tc-u 0>r)-JC_L X< lc-- UI 0. o o UI -- Z z o *s, r-- ,0, 0,0, 0,0 Cc Oc 10 C1D C+M O i--+ | CM in cn ri+n- cO+o X 0000 COr-- COCO -- -O O 0) i~ +> +> (0 C 0) r- "O O 0) 1- 43 43 f0 I= QJ r-- O O 01 S_ 43 43 (0 C 0) 43 Cl) <Uu Xe) CcOunL ioe- 3C -+awur>t- 4- -cO 43 c aj tA u Q) <D tA a Vs: z H * > ul _l 00 h<~c ari ou -a 4O3J f4O1--3-(--0<u0) co oao t- T4O33l I- 10 4OL3 l0--U) Co a u af- *4t-ou f4--soc-->hufa-O> oo oao h 4U031 4< a 0u1 3to ao. D0C1 <CuJ 01 <3CvJ JWklC OJ C E LX CM 0 CO o s- o t- O - ti <U O 1-- O h- l-- ll- s- 440-3) 0s)- i001Qa -0aX)ih- 3l/l UoCXJL s "O I--> it0c-)o 4 J CL (OcCS --C +- t/J 303 TABUE V. SERUM IMMUNOGLOBULINS IN MICE AFTER A SINGLE DOSE OF 10 ng/kg TCDD Group Control Treated Serum Ig Levels3 After 2 weeks 4 weeks 8 weeks 1.37+0.23 1.13+0.10 1.24+0.07 1.13+0.19 1.20+0.13 1.09+0.16 3Values are ratios of total serum immunoglobulin to that of a pooled mouse serum sample. Mean + SE of 3-4 observations per group. The severity of pathologic changes in the liver was most pronounced at 4 wk after treatment. At 8 wk liver lesions were more of a chronic type, i.e. fibrosis. The incidence of thymic changes was highest at 4 wk post treatment and even though a decreased thymus wt was noted after 8 wk, histologic changes were not observed at this interval. In another study where mice were given repeated weekly doses of different amounts of TCDD no effects on the thymus after 8 wk were noted (13). These findings indi cate that the toxic effects of TCDD to this organ are not only reversible but even with continued exposure there is an adaptation. The single dose oral LD50 of TCDD in male mice is reported to be 114 ng/kg (18). Following a single administration more than half the total body burden for TCDD is accounted for in the liver in a sensitive strain of mouse (9). According to the data presented by these authors, the con centration of TCDD from C5 7BL/6J mice liver disappeared at a half-life rate of approximately one wk. This value is considerably smaller than the whole body half-life value obtained in rats (8,11). In view of these ob servations a major fraction of TCDD in the liver of mouse may have been eliminated in 8 wk, while it may be more persistent in other species, e.g. rats. If this interpretation is correct, the effects of TCDD in liver and other organs will disappear in this time interval. A marked effect of TCDD exposure was seen on the splenic lymphocyte transformation in cultures. A single dose of TCDD caused a considerable increase in spontaneous blast formation in splenic cultures obtained from treated animals. This finding was also observed when exposure of TCDD was repeated once a week for 8 wk (13). Although the implication of this find ing is not clear, nor is its mechanism understood currently, this effect is most striking 2 wk after administration of 10 yg TCDD/kg. After 4 wk the effect persisted, although there was great individual variation in these parameters. In another study (data not reported) we exposed adult (4-5 mo old) mice to the same dose of TCDD (vs. growing mice in present report) and a similar, although less extensive, effect was seen when the spleens were cultured 2 wk after treatment. Another important consideration of this increase in spontaneous lymphocyte transformation is decreased respon siveness to mitogenic agents giving rise to smaller stimulation indices in treated animals. This is indicative of decreased responsiveness of the immune system to antigenic stimulation, also confirmed in another study (13). The major finding of this study is that both spontaneous blast for mation and mitogen induced stimulation return to normal in 8 wk after TCDD exposure. This further supports the argument of the reversal of toxic ef fects of TCDD in mice within a relatively short time. It is difficult at present to extend these findings to an accidental human-exposure to ICDO__since it is well known that considerable species variation occurs in response to this toxic chemical. But the differences in physiological disposition would alter the rate at'which the body is cleared of the chemical and if a sublethal exposure occurs, the effects v probably can be reversed with time. The immunologic effects were not last- 304 ing whereas the hepatic effects persisted. Perhaps accumulation of TCDD in liver may be considered a defensive mechanism, at least insofar as the immunologic alterations are concerned. ACKNOWLEDGMENTS ^ The valuable assistance of Roselle Lisowe and Ruta Kalnins during these studies is gratefully appreciated. LITERATURE CITED 1. Bjerrum, 0. J . , A. Ingfld, H. Lowenstein and B. Weeke. 1973. Carba- mylated antibodies used for quantitation of human IgG. A routine method. In: Quantitative Im m unoelectrophoresis. N, H. Axel Sen, J. Kroll and B. Weeko, Eds., Universitetsforlaget, Oslo, pp. 145-148. 2. Buu-Hoi, N. P., P. H. Chann and G. Sesque. 1972. Organs as targets of dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) intoxication. N a tu r w is s en s c h a fte n 59:174-175. 3. Courtney, K. D. and J. A. Moore. 1971. Teratology studies with 2,4, 5-trichlorophenoxyacetic acid and 2,3,7,8-tetrachlorodibenzo-p-dioxin. T o x i c o l . Appl. Pharm. 20:396-403. 4. Elvidge, D. A. 1971. The gas chromatographic determination of 2,3, 7.8- tetrachlorodibenzo-p-dioxin in 2,4,5-trichlorophenoxy acetic'acid and 2,4,5-T esters and 2,4,5-trichlorophenol. A n a ly s t 96:721-727. 5. Gupta, B. N. , 0. G. Vos, J. A. Moore, J. G. Zinkl and B. C. Bullock. 1973. Pathologic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in laboratory animals. Bnv. f i l t h . Pers. 5:125-140. 6. Harris, M. W . , J. A. Moore, J. G. Vos and B. N. Gupta. 1972. Gener al biological effects of TCDD in laboratory animals. Env. m t h . Pcrs. 5:101-110. 7. Kociba, R. J., P. A. Keeler, C.~N. Park and P. J. Gehring. 1976. 2 .3.7.8- Tetrachlorodibenzo-p-dioxin (TCDD): Results of a 13-week oral toxicity Study in rats. T o x i c o l . Appl. Pharm. 35:553-574. 8. Piper, W. N., J. Q. Rose and P. J. Gehring. 1973. Excretion and tis sue distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat. Env. f i l t h . P ers . 5:241-244. 9. Poland, A. and E. Glover. 1976. Stereospecific, high affinity bind ing of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol, j . D io l. Chem. 251:4936-4946. 10. Poland, A. and A. Kende. 1976. 2,3,7,8-Tetrachlorodibenzo-p-dioxin: environmental contaminant and molecular probe. Fed. proc. 35:24042411. 11. Rose, J. Q., 0. C. Ramsey, T. H. Wentzler, R. A. Hummel and P. J. Gehring. 1976. The fate of 2,3-,7,8-tetrachlorodibenzo-p-dioxin fol lowing single and repeated oral doses to the rat. Toxicol. Appl. Pharm. 36:209-226. 12. Schwetz, B. A., J. M. Norris, G. L. Sparschu, V. K. Rowe, P. J. Gehring, J. L. Emerson and C. G. Gerbig. 1973. Toxicology of chlo rinated dibenzo-p-dioxins. Env. H ith. Pers. 5:87-99. 13. Sharma, R. P, and P. J. Gehring. 1979. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on splenic lymphocyte transformation in mice after single and repeated exposures. Ann. n . y . Acad. s c i . (In press). 14. Steel, R. G. D. and J. H. Torrie. 1960. P r i n c i p l e s and Procedures o f s t a t i s t i c s , McGraw-Hill, New York. 15. Thigpen, J. E . , R. E. Faith, E. E. McConnell and J. A. Moore. 1975. Increased susceptibility to bacterial infection as a sequela of expo sure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Infect:, immun. 12:1319- 1324. 16. Vos, J. G. and J. A. Moore. 1974. Suppression of c e T V U r immunity 305 1n rats and mice by maternal treatment with 2,3,7,8-tetrachiorodibenzo^p-dioxin. i n t . Arch. A l l e r g y 47:777-794. 17. Vos, J. G., J. A. Moore and J. G. Zinkl, 1973. Effect of 2,3,7,8tetrachlorodibenzo-p-dioxin on the immune system of laboratory ani mals. Env. H lth . P ers . 5:149-162. 18. Vos, J. G., J. A. Moore and J. G. Zinkl. 1974. Toxicity of 2,3,7,' tetrachlorodibenzo-p-dioxin (TCDD) in C57B1/6 mice. T o x i c o l . Appi, phjrm. 29:229-241. 19. Woolson, E. A., R. F. Thomas and P. D. J. Ensor. 1972. Survey of polychlorodibenzo-p-dioxin content in selected pesticides, j . Ay. Food Chem, 20:351-354. 20. Zinkl, J. G., J. G. Vos, J. A. Koore and B. N. Gupta. 1973. Hemato logic and clinical chemistry effects of 2,3,7,8-tetrachlorodibenzop-dioxin in laboratory animals. Env. H lth . P ers. 5:111-118. ij: i, in 1 _ \ 306y V EN V IR O N M EN TA L PO LLU TIO N