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* r. r il < ! % t t i Jt. ' * .i 5!' : t. i-ty"* ,,* ...Wyif _ Vs' jL?r > I------ ^ 'yfcU J Premature Births in California Sea Lions: Association with \ peaks on the gas chromatogram High Organochlorine Pollutant Residue Levels tained with a DC-200 column; q fication was achieved through a parisoa of the height of the peak c Abstract. Prematura pupping in California iea lions has been noted on the ing between the peaks for p,p`- breeding islands since 1968. Organochlorine pesticides and polychlorinated ind p.p'-DDT with the height 1 biphenyl residues were two to eight times higher in (issuer of premature parturient corresponding peak in chromato females and pups than in similar tissues of full-term parturient females and pups of the standard (Arodor 1254, collected on San Miguel Island in 1970. santo Co.) (6). Blubber aod liver The California sea lion (Zalophus ealifornianus calijorniunus) breeds OH California's Channel Islands and on islauds off the Pacific and Gulf coasts of Baja California, Mexico. In the Chaooel Islands normal pupping occurs between 15 Msy end 30 June and the end of premature births and the onset of the normal pupping season. About 15 May the death rate decreases and some pups survive. In mid-May both live premature and full-term pups are present; full-term pups are easily distinguished as they have full motor from females were analyzed as represent a storage and metabolitr for organochlorine residues. Brai sue from pups was analyzed to some insight into the behavior o premature pups. Organochlorioe cidt and PCB residues uere iou; all tiasucs collected and tested ( breeding between 20 June and 20 July. coordination. The quantitative incidence 1). The must prevalent neuboliu We have observed esrly termination of of premature births has not been well found to be DDE 180 to 93 perce pregnancies amoog animals on several breeding rookeries, including those on Sen Miguel Island, since 1966. Prema ture births were reported by Simpson and Qilmartin (J) on San Miguel Island and by Odell (2) on San Nicolas Island, established. Oo San Miguel Island we counted 242 dead premature pups on 25 April 1970 and 348 on 18 May 1971 from an estimated total popula tion of 10,000 to 15,000. On San Nicolas Island, Odell (2) reported 442 the total DDT). All analyzed tissues from fei which gave birth prematurely in contained higher concentrations the tissues from females which birth to full-term pups. The mean and were discussed by Brownell and premature pups from a female popula DDT residues in the blubber and LeBoeuf (5). An earlier investigation tion of 5,500 between 17 January and of premature parturient females into the causes of these premature 3 May 1970. 8.0 and 3.8 times greater, rtspecti births was inconclusive (/). The work In 1970 we collected two groups of than the concentrations in the reported here was conducted to deter postpartum female California sea lions tissues of normal parturient cows, mine whether environmental pollutaot within 24 hours after parturition on brains of premature pups cont residues may be related (o premature San Miguel Island. The premature twice the residue concentrations a partus in the California sea Hon. parturient group of six females and brains of full-term pups. The In the Channel Islands premature their premature pups were collected be levels in blubber, liver, and brain o births occur as early as January and ap tween 20 and 24 April. The full-terra premature parturient group were pear with Increasing frequency until parturient group of four females and 4.4, and 2.4 times higher than t mid-May. We observed live premature their live pups were collected between in the full-term group. In ne pups from February through mid-May, 24 and 26 June, late in the normal blubber nor liver did the ranges of and saw no pups which were dead at pupping season. due values of total DDT or PC birth. The early pups are not furred Ages of adult females were deter premature and full-term groups < and appear to die soon after birth. The premature pups born in March, April, and early May are furred, but they lack motor coordination, their breathing it short, and only portions of the lungs have been aerated. Late premature pups mined by counting growth layers in the dentine of longitudinally sectioned canines (4). The liver, brain, and blubber from cows and pups were ana lyzed for DDT compounds (p,p'-DDT, p.p'-DDD, and p.p'-DDE), dieidrin, lap (Table I). Dieidrin residues not detected to all samples and \ present, were low. Figure l it a h gram of the DDT and PCB reside the two groups of animals, showing relative differences in tiue polh appear 10 live from a few hours to and polychlorinated biphenyls (PCB's) concentrations. possibly as long as several days. There (5). Identification of PCB's was Histological sections of liver ti is no apparent temporal break between based on the characteristic profiles of revealed norma) tissues from all prt Table I. Concentration of pollutant residues is tissues of parturient California see lion females and pups, in parts per million (ppm', weight; N, number of animals; JT, mean of two (brain, liver) end three (blubber) midue determination, on ea?h tmue of each sn The age range Is 6 to 12 years for prematura parturient females and to to IS years for fuJI-teira parturient femaJes. The numbers in p. theses are standard deviations. Tissue Blubber (female) Brain (pup) Blubber (female) Liver ((erMle) Brain (pup) Mean age (/ears) 6e 3I 6 4 12 4 12 4 F(%> DDT (total) Mean Range T (ppm) Range (ppm) 84 1.7 3.3 Prtmafura 77-87 t.5-2.2 3.2-3.4 824.4 (167.S) 25.24 (4.1) 2.38 (1.27) 626-1039 22-30 0.82-4.15 Futf-term 85 84-88 103.2 <69.7> 51-203 4.4 2.9-5 5 4.8 4.6-J.l 6.67 (5.1) 1.20 (1.10) 2.4-13.6 0.36-2.82 PCB* X . (ppm) Ra (PI 112.4 (2441 5.74 (1.45) 0.45 (0.32) S'',4. 0.23- 17.1 (6.1) 1.32(0.76) 0.1** 10.05) 12 0 52Di2 MOMS 08.U63 Lr# partus females except one. The L-er of ibis animal showed a few midVjbiI foci of mononuclear and polyftojphonuclear cell infiltrates. These ,boormal findings apparently ate not (imilar to histological changes in rats d organochlorine pesticide. (7) or jdeoU fed PCB compounds (4). Sero>gicai analysis revealed no antibody iltrs to Leptospira species or Brucella ,bortut in the six cows of the pruneire parturient group. Bacteriological islysis of the uteri of this group yielded isolation of Escherichia coll {root two of the six. Liver mercury residues (wet weight) | b the three premature parturient fe males ranged from 38 to 64 parts per ' , aillion (ppm) and in their pupa ranged rom 0.4 to 1.8 ppm. None of the tisuet from full-term animals were anayxtd for mercury. The difference between the conceitiittona of organochlorine pesticide eod PCB residues in the premature end ull-term groups indicates e possible ause and effect relationship between ugh levels of these pollutants and early lamination of pregnaocy in California ea liona. High concentrations of or^nochlorine residues are implicated in icproductive failure in ranch mink fed Ssh from Like Michigan (9). Experi mental feeding of PCB compounds bu lean shown to cause reproductive failno and death In adult ranch mink 110). There it also experimental eviIsnee that p^'-DDT induces premature parturition in rabbits (if). Some orpoocblorino insecticides and PCB impounds induce hepatic microsomal oxyme activity resulting in increased etebolism of progesteroae end estra diol In pigeon livers (12). Similar entyme induction has been demonstrated io the rat (7). Definitive knowledge of the combined toxic or synergistic effects sf organochlorine pesticides, PCB's, tad mercury is lacking. LeBoeuf and * Bonnall </i) reported high residue kvsli from three "apparently healthy" (smalt California sea lions collected in September 1970 at San Miguel Island. Apparent good health of the female does not exclude t history of premature parturition. Since they did not report the reproductive histories of those aniosli, it is impossible to relate their find ings to the premature parturition phenomena. Tha exact relationship of pollutants 0 premature births aud the media `aisms involved can only be elucidated through laboratory experiments. Such Experiments with California sea lions Fig. I. Histogram of mean concentrations of total DDT and PCB's io tissues of Cal ifornia sea Hons delivering prematurely (cross-hatching) and at full term (stip pling). have not been possible because of tbe cost of maintaining large breeding col onies of sea lions in captivity. However, considering the magnitude of the differ ences in residue levels found in the two groups sampled from the wild popula tion, we feel that the possible ceuse and effect relationship cannot be ignored. A possible explanation for the differ ence in pollutant residue levels in the tissues from the two groups of sea liona is utilization of different feeding arena. It has long been known that after the breeding season male California sea lions move northward. It it believed by some that many females of the Channel Islands population move south and win ter in waters off southern Beja Cali fornia (14), while e portion remain in the general area of the Channel Islands. Organochlorine residues in a marine crab (IS), marine fish (16), and the . brown pelican (17) are higher around Los Angeles, California, than in Baja California or in northern. California. Thus, female sea lions wintering in the southern areas would be feeding on fish having smaller amounts of organocblorine pesticides and would therefore assimilate smaller quantities of the pesticides than females wintering in the Channel Islands area. Tbe mean age of premature parturi ent females is less than that of fullterm females (Table 1), but the age ranges of the groups overlap. Because of the smalt sample sixes and the overiap in age ranges it is not possible at this time to relate the observed differ ence in organochlorine residues or tbe premature parturition phenomena to age. We think it is uoiikely that the ob It SBPTBMHR ItTS served difference in organochlorine resi due concentrations could be accounted for by the premature parturient females being collected 2 months before the full-term females. Five sea lion females collected randomly from the San Miguel Island population in July 1969 had total DDT metabolite residues in the blubber ranging from 17 to 98$ ppm (/$), which indicates that some females can be. expected to have high residues el any time of year and that a profile of the population probably ranges from animals with low to those with very high concentrations of organochlorine residues. Unfortunately, nothing is known about the fat dynamics in ges- taring female sea lions or about changes ia organochlorine residue storage de pendent on hormone cycles. Net mo. biiizstion of fat to meet increasing energy demands would be expected to increase residue levels in the blubber, yet we found that the full-term females had lower organochlorine residue con centrations. Robert L. DbLono* Marine Mammal Division, National Marine Fisheries Service, Seattle, Washington 98113 William G. Gilmartin John G. SimpsonI Naval Undersea Center, BlO`Systems Program, San Diego, California 92132 eftieaces tod IMm t. J. O. Simpson end W. Q. OilmarUe. meScirntt 3*. 2l (1970). 3. D. K. OdelL in Proceeding* ti *A* 7th Annuel Btonnar amt Dhlng Mammal Canftranca (Stanford Swirdi Institute, Menlo Peek. Calif.. 1970). J. R. L, Brownell end B. 1. LeBeeef, In Jiehtlcal end OceenogrepAkef Servo et the Sam Swim Chmmet Otf Spilt. INI* 1970, D. Siraughtn, Ed. (UntvenRr of Souibent Csliforoie, Loe Angeles. 1971), vo). t, pp. 2S7JOS. 4 V. B. Scheffer, Setence IIS. MS (1990): R. M. Lews, Nature 1*9. 972 (1992). 5 The DDT compound, wet* p.p'.DDT (1.1.1. mth!oro-2.Z-bi!<P<hloT*n'htGj!)hM>e), p,pDDD [1. |^tcMer2.2.bU(p<)i>oropaen>l)ta. ne), and e.it'-DDI ll,1-4<cHtoro-3,2-Mg<*. chlorophenyl)ethylcne|. The analytes for these compounds, dtsldrin, end PCB's were made by the Wisconsin Alumni Research Founds, tion, Msdlson, according to the ftandardlssd techniques liven In: Food end Drag Admin istration. Frillc/dt Analytical Manual (Depart ment of Health, Bdwration, and Welfare. Washington. D.C.. 1M5-196I). vol. I. looseleaf. 6. D. W. Anderson, J. I. Hickey. R. W. Ritebrough, D. P. Huahes, R. C. Christensen. Can FlalA Natur. SI, g (1969). 7. L. O. Hart end I. R. Pouts, NaunynSehmlaSthartt Arch. Sxp. PoiAef. PAameAol. 149. 416 (|P65). 9. J. W. Miller. Pu*. Maalik Bee. 99. 1095 (1944). 9. R. 3. Aulerich, R. X. Ringer, H. L. Scegnn, W. O. Youatt. Can. J. Zeal. 49, 611 (1*71). 10. R. X. Ringer, R. 3. Aulerleb, M. ZaMk, In preprint ot papers presented the I64lh national meeitnt of the Amerisen Cbemleal Society, Division of Water. Air, and Watte Chemistry. New Yotk, 21 August to 1 Sep tember 1972. pp. 149-1S4. 11. M. M. Hart. R. H. Adamson, S. Fshro. Arch, tnt. FharmntaSm. Thar. I9S, 216 (1971). 1169 lio n s 0 flil6 4 v. t-* ; ;.<.:* 4-v*.' 1 *' 1'; **' .'* jAjWMfc^MtoAiwBfcliniil dWi** , :?r1/ w. 12. D. B. Fa*ka. Nat lit. SOS 09*7); R. W. RiMbr*ii*h. P. Hatch*. D, realtatl. S. O. Harman, M. V. Kirvcn, IM. MO, Kl (1961). 1). B. J. LeBotuf Hitt M. L, Bonncll. Ihtd. 254. too (ItTI). 14. O. A, Bartholomew, in Froeaedlnfi of the Symposium on the Blolofy ot the CaU/onis ItlpnOt, K. -N, Phtlbriek, Ett. (Suu Barbara Botanic Gardens, Santa Barbara, Calif.. 1947). 15. H. Burnttt, Science 174, <0* (1971). 16. R, \V. Riwbruttiti. D. B. Met)re), D. i. Martin. H. S. Oicoit, Fettle. Monit. /., in prtu. )7. X. W, RtMbroush, in troesoiints of the Sixth Berkeley Symposium on Mpthematiet, Statistics, tmd PmhohlUty (Unit. of California Ptew, Brkl". 1912). pp. 44S-46). It. R. E. Anai, U S. Dtp. Commtrct Spoc. Scl. Hep. Fish. No. 62S (1971), p. 29. 19. Wa thank A. M. Johnson anti R. W, Rise- broujh for aiding in Iha dailan antt Inter pretation of h* study and It. AnUindli, M. Dailey, S. Leatherwoott, antt B. Stronte (or aiMsn:e In rapturing tha mo Horn * Present address: School of Wildlife antt Fish* odea. Department of Fortuty antt Coastrva- tlon, Unlvanlty of California. Berkeley 94720. t Praaant acittrcM: 1972 Newbury Hoad, New bury Park. Calif. 91120. 16 October 1972; revised 30 January 1973 a Cell-Mediated Immune Responses in vitro: Interaction of ThymusDerived Cells during Cytotoxic Allograft Responses in vitro Abstract. A mouso in vitro allograft system was used to cheek for synergism among thymus-derived cells during ce//-mrfiafed immune responses. Synergistic Interactions occurred between thymus and thymus-derived lymphocytes, The majority of precursors of cytotoxic effector cells were peripheral thymus-derived ceils; thymocytes acted mainly as amplifier cells. Both murine thymocyte* end thymusderived (T) lymphocytes have been shown to be capeble of mounting cyto toxic allograft responses in vivo (/) and in vitro (2), apparently without requiring collaboration with bone mar row (BHJerived lymphocytes. When compared on a cell to ceil basis, thymo cytes were found to be poor precursor cells of cytotoxic lymphocytes (CL) in vitro in contrast to recirculating T cells, splenic T cells being intermediate (2). These findings suggested that there exists a heterogeneity within thymusprocessed lymphocytes in their capac ity to differentiate in vitro into cyto toxic effector cells. Indeed evidence for the existence of functional T cell sub populations has appeared (J, 4). For example. Cantor and Asofsky (4) pro posed a model for the generation of thymus-derived effector cells mediating graft-versus-host reactions, in which two distinct types of T cells appear to be involved: one acting as an amplifier cell and the other type functioning as the precursor of the effector cell. I have now tested the concept of T-T cell interactions during cell-mediating im mune responses in an in vitro allograft system. About 15x10* viable dissociated CBA/H/Wehi (H-2*) or AKR/J (H2k) mouse thymocytes or inguinal Table I. Synergistic effect of a mixture of thymocytes plus syngeneic lymph node cells in cytotoxic aUogftft responses in vitro. Test system Lysis (%)* of "Ct-libcM PUS targel cells (H-2*) at a CL to target cells rttio of: 12 : 1 2 : 11 0.3 : l 13 x 10* CBA LN 13 X 10* CBA LN, treated w)ih antiserum lo d IS x 10* CBA thymocytes 1.3 X 10* CBA LN phis 14 X 10* CBA irrtdiaietl thymotyw* 1.3 x tO* CBA LN plus ttt X 10* CBA thymocytes t.S x 10* (CBA LN), treated with antiserum to plus 14 x 10* CBA thymocytes 1.3 X 10* (CBA X BALB/c) F, LN plus 14 X 10* CBA thymocytes J.3 X 10* CBA LN plus 14 X 10* (CBA X BALB/c) F, thymocytes 1.3 x 10* (CBA I.N) mitomycin C-treated plus J4 x >0* CBA thymocytes 100 80 40 13 12 12 30 14 12 25 13 12 100 91 52 23 14 12 2) 13 12 38 27 13 24 12 12 Ktkfiondrr kIJi (H-2) were cultured (utUir with 2 X10* mitomycin C-lreMcd allogeneic ALB/c (H-2) spleen cell*. Cytotoxic activity gentreted In vitro wai essayed in a Cr tew for 200 minutes. Cytotoxicity obtained wot compared on a culture bails relative to the cytotoxicity ((iterated by CIIA LN cell derived cytotoxic lymphocytes. Background lysis ol P*15 target ceil* (in the presence ol mmal lymphocytes) wi, 12=1.3 percent. Similar results were obtained in 12 indertnttem expenmtnts. lymph node (LN) cells, or a t of both, were cultured togethe 2x10* mitomycin C-(reated B. spleen cells (H-2J) for 6 days in Tied Eagle's medium containing cent fetal calf serum in the Mar Diener culture system (2, 5). from three cultures per group pooled, washed twice, and assay' in vitro generated cytotoxic actis a modification (2) of the *>Cr r assay described by Brunner et a Cytotoxic activity generated in vitt compared on a culture basis (7). the number of lymphocytes at the ation of the culture was kept id* in the various groups of cultures the cytotoxic activity generated compared relative to that obtained LN cells as precursor cells of cyu lymphocytes. In actual fact, cyu lymphocytes harvested from dift groups were assayed for cytotoxi livity at the cell concentration v resulted (in the case of LN de cytotoxic lymphocytes) in a rati cytotoxic lymphocytes to target of 12 to !, 2 to I, and 0.3 to l. DBA/2 derived mastocytoma cell P815 (8), cultured in vitro an* identical H-2 specificity as BA! spleen cells used for immunization, used as target cells in the 9,Cr re assay. AKR antiserum to CBA 0 gen was prepared according to (9) and used as described (7). antiserum killed 80 percent of ( thoracic duct lymphocytes io the ; ence of agarose-absorbed guinea serum, and the activity against 9 c be absorbed by brain from CBA n Mitomycin C treatment was adm tered at a final concentration of Hg/ml for 30 minutes <2). Irradia (900 rads) of dispersed lymphot was performed as described by M and Sprent (10). As given in Table 1, CBA LN t (an example of a lymphocyte pop tion containing 80 percent of periphi T cells) represented a good precui cell population for cytotoxic lymp cylev. Thus, after a 6-dsy culture pei in the presence of cell-bound i antigen (2x10* mitomycin C-tre;. BALB/c spleen cells), cytotoxic aefiwas generated which lysed 80 perc of the blCr-]abe!ed P815 target c at a ratio of cytotoxic lymphocytes target cells of 2 to 1 within 200 rr utes. That the reacting cells were lymphocytes was suggested by (he f that treatment of LN cells before c lure with AKR antiserum to 0 anticplus complement, abolished the cap ily of the cells to movtot a cyioto- liter ~ HONS 0Q2165