Document 8RKmwvXJ2GK4Qo360NNvRj4ya

... Sex Diiloicnee in the Number of Adipose Cells from Genetically ....Obese Rats.. (t i< well known that there is no increase in the. number of '"adipose cells in many kinds of cx|>crimenlal obesity. In ` * f,enetiiaHy obese mice, obob obese hypcrglycacmic1*2 or yellow als-rac5 xt'juiiraucx -a* well ns in bypotlialamic ol>eitlies produced 4-vtsy**M*u*hfaFhcwe--s,n luice4 or stcrcotaxw'>1csions in rats5, -`.V-aliorr. enordy xvtLiTgemcnt of the adipose'tells. The same > - -rerulis arc obtained in rats made obese by "a high fat diet''. There is only one known exception to this rule: obesity induced by a high fat diet in female mice7, in which the number of cells is increased by a factor of 2.5. because male animals A-me used in ail experiments except the last, the question arises whether there may be a sex difference in the adipose tissue development in obese animals. iicncticolly obese rats ffafa)* 2.5 months old and their filter males, fed with my control diet, were used. Whole 'prrijienilal fat pads were weighed and treated as previously vitostTibcd6'.' portkvin'of histeHogicai w*Uoiii *cm slides, pro- '`gaoled grownvT-idv.M of u/l*iojoituva' 'Sw.Tosicope, were juasnlBd for lhcir.aflipr>sc ocils at a magnifientmoof xJ30. The - mean volume was calculated and the number ~of adipose cells estimated by the ratio of fat pad weight to mean adipose cell .volume-*4X91 (0.9.1 is ihc measured density of the pads). '.TnWc 1 .shorn! haf the. body weights of obese rats doubled ..-so. the iconics hut increased less in the males. The weight of pcrigcsulal fat pads increased 3.2 and 9.7-fold in males and fannies respectively. Adipose cells enlarged 4.6 and 4.0- fold in obese males and females respectively. In male obese rats there were significantly fewer cpididymal adipose cells than in their litter mates, but a 2.3-fold inctcasc in their number t vjawcofacarvnd i4ha^uttiructrin! cells of Ihc obese-females. X*hk> 1 Weiohts of tlio Body and of the Two Parir,enital Fat Pods of .JJk Month Old Gonoticnlly Oboso Mala and Female Rots and Thoir . Littor Matos Lean litter males Obese Males .Body weight (g) 276 16.0 Fpididymal fat pad weight (g) 2.82 0.260 ift ttiHfimr nnllweduae iyt KF pm5) 276 25.5 ...jymiarfa*li|sretc4Jls<a4sllcu>s) >#34 0.458 366+17.3 9.00 0.327 1,275 85.2 ;* &50 0.256 Females Hedy-weight (g) ' !65 2.4 .-JlowwcSfiat fm-pad wctgha (g) 4.64 0:1*9 ' -^Mspaao-aeM volume 4 x'WF pm*) ,r379'46.6 adipose cclh (nsidioati) v *4.08 0.292 ' T20 13.6 ' ' 15.9 0.71 "TI,536 43.5 'v"' ' 9.48 0.460 Ttc cell volume of the obese groups increased, but celt number increased only in the female group (2.3 limes). All differences .are. highly significant (/'<0.01). Values arc mean s.e. There -were.six animats in each of the four groups. -:-l(y**er<rophy etf thc adipose cells in the olme Jcmales could only account for a parametria I fat pad weight of 1.64x4.0 = jt|iM g--9.26 s less dun thp actual weight. Audio about 70% of4iic weight of the pads was due to formation of new fat cells which w-cjc themselves hypertrophied, i These results clearly established a sex difference in live ; .development of pcrigcnhal .adipose tissue. They.arc in good : agreement with the literature which records no increase in fat sell number in male obese rats or mice1 *6. But there was a (parked hyperplasia in the p.iramclriul adipose cells in the | (empales which was very similar lo those observed in female mice , njadc obese by u high fat diet7. . .. . NATURE VOL. 231 MAY 7 1971 ' Exceptions to the rule that only hypertrophy is observed in male adult animals had been claimed: there is hyperplasia of adipose cells in rais after repeated injections of insulin"-"' or meal feeds". In these animals there was no increase in the weight of the cpididymal fat pad, and adipose cell volume seemed to be reduced; the differences, however, were very small. The principal effect, in the two eases, was a great increase in the total DNA content of the pads (+ 50% for meal fed rats" and + 100% for rats treated will* insulin10). Such differences cannot be the effect of n marked hyperplasia: ihc weight of fat pads was unchanged and cell volume very slightly smaller; so it can be concluded that there was an increased DNA content in stromal structures. Such on effect has been established in hypophyscclomizcd rats after injections of somatotrophic hormone: the rate of DNA synthesis of supporting and vascular structures was enhanced, but there was no effect on division in adipose cells". In my rats, the histological appearance of pcrigcmial adipose tissue was (he same as that described for rats made obese by a high fat diet4. There was no difference, except for the size of Ihc cells, between fat tissues of control and obese rats, although there was a marked and similar hyperinsulinism in males as in females in the obese group (to be published later). 1 thank A. Alexia for technical assistance. Daniel Lemonnier Laboratory of Biology and Human Nutrition, CNAM 292 Rue Saint Martin, 75 Paris 3 _ Received December 15, 1970. 1 Heilman. D.. Tiiljcdal, 1-13., and Westman, S,, Ada Morphol. Neert-Scand., 5. 182 (1962). * Lemonnier, D.. Winand, J., Furncllc, J., and Christophe, J. (in the press). ` 5 Heilman, R., Thclandcr, L., and Tiiljcdal, I-B., Acta Anar., 55, 286 (1963). 4 Heilman, 11., Tiiljcdal, I-B., and Pelcrsson, B., Med. Exp 6, 402(1962). * Hirsch, J., and Han, P. \V., J. Lipid Res., 10, 77 (1969). * Lemonnier, D., Arch. Anar, Microsr. Morphol. Exp., 59, 1 (1970). 1 Lemonnier, D., Experientia, 26, 974 (1970). *. Zuckcr, L. M., and Zueker, T. F., J. tiered., 52, 275 (1961). * Hausberger, F. X., and Hausberger, B. C., Anat, Ree., 127, 305 (1957). . * Kazdova, L., and Vrnna, A., Norm. Metah. Res., 2. 117 (1970). 11 Braun, T., Kar.dova, L., Fabry, P., Lojda, Z., and ITromadkova, V.. Metabolism, 17, 825 (1968). 11 Hotlcnbcrg, C. H,, and Vest, A., J. Clin. Invest., 47, 2485 (1968). Polychlorinated Biphenyl absorbed from Sediments by Fiddler Crabs and Pink Shrimp . Polychlorinated biphenyls (PCBs) arc manufactured in the United Slates, Europe, and Japan and used as plasticizers, flame retardants, insulating and heat exchange fluids and in many other products'. Structurally related to DDT, soluble in lipid but relatively insoluble in water, and extremely persis tent in the environment2, they have been reported in many marine and estuarine organisms5. One PCD, 'Arcelor 1254' (Monsanto), was reported in water, sediments, and biota from Escambia Bay, Florida4. In August 1969, pink, while and brown shrimps (Penaeus duorarurn, P. setifvrus, and P. aztecus) from Escambia Ray were found to contain whole body residues of `Aroclor 1254* at concentrations as high as 14.0 p.p.in. Most was concen trated in the hcpatopancrcns; residues in seven composite samples of at least five shrimps ranged from 0.6 to t?n.o p.p.m. In April 1970, fiddler cruhs ((tea minnx) collected at three stations along the lower Escambia River and upper Escambia Bay had individual whole body residues of 0.45 to 1.5 p.p.m. DSW 0 3 2 8 8 3 * 1 0 . , STLCOPCB4016845 NATURE VOL 231 MAY 7 1971 Cl J To determine lire distribution of `Aroclor 1254' in the upper end of five aquaria inclined 10 degrees so ns to ,,- estuary, foil)1 samples of water were collected in glass bottles; a natural environment. Ten crabs from a population winch (wenly sediments were collected with a core or drcd(:o sampler, and many estuarine organisms were captured by trawl and drcd|:c. These wert analysed by gas liquid chromatography4. Tlte largest aeaumtlulions of 'Aroclor' were found in the had no contamination were placed in each aquarium wuh on= serving as a control. Water with an average salinity (,f 27 j, p ( at 13 C (lowed at 5 l./h through the lower end. Holh shrimp/ and crabs were fed lisli containing <0.0.1 p.p.m. 'Aroclor sediments. In one survey (February 1970). concentrations 1254' each day for 30 days, l ive crabs were removed from ranged from 0.6 to 61.0 p.p.m. in a sample taken at the dis- each aquarium, killed and rinsed with a 50% mixture of cltarg.c point of the industrial plant that accidentally released acetone and water to remove `Aroclor 1254' from material Ihc chemical and at eleven stations extending 16 km down* adhering to tlte cxoskclclon. To ensure enough tissue for stream (Fig. I). `Aroclor' was not detected in sediments analyses, individual crabs and pooled samples of hepato- collected above the plant but soil samples from the bank near pancrcas from shrimp were mixed with anhydrous Na2S04 and Ihc mouth of the river 6.5 km downstream from the source had extracted with petroleum ether in a Soxhlct apparatus; the 1.4 to 1.7 p.p.m. of this chemical. Subsequent samplings from extract was purified with `Florisil'5. `Aroclor 1254' was three stations in the Day showed little change in the amounts of quantitated by gas liquid chromatography4. The ratio of the chemical even after nine months. We therefore investi individual 'Aroclor' isomers maintained integrity in tlte gated wlrclhcr shrimp and fiddler crabs could accumulate sediments and tissues of test animals throughout the investi t `Aroclor 1254' from the sediments. gation. i Table 1 `Aroclor 1254' in Individual Fiddlor Crabs and Mepatopancreaj from Pink Shrimp Sediment `Aroclor 1254' Average and `Aroclor 1254' in in sediment range of `Aroclor combined hep- (p.p.m. dry 1254' in five atopancrcns from weight) individual fiddler four or more crabs (p.p.m. shrimp (p.p.m. wet weight) wet weight) Sandy sill * ' Silt t Sandy sill * Silt T ' . Sandy silt* Silt t Silt t Sand control* Silt control t 61.0 30.0 5.7 4.9 4.1 2.5 1.4 ND ND $0.0+25.0 17.0 9.0 -- 3.60.9 -- 3.2 0.9 ------ 0.3 0.2 -- 240.0 6.1 9.8 1.3 6.7 l.l 0.2 <0.2 <0.2 The samples of crab and shrimp were kept for 10 days on naturally occurring sediments containing 'Aroclor'. Mortalities occurred in sonic groups of shrimp which could not be attributed to 'Aro clor 1254". ' Sediment which contained particles of sand > 354 pm in diameter. t Sediment which contained particles of sand 154 pm or less. --, No sample. ND, Not delectable. KILOMETERS Fig. 1 Study area showing lower Escambia River, upper Escambia Hay and stations where sediment was collected on February 24, 1970. The amount of 'Aroclor 1254' found at each station is given in parts per million (p.p.m.). ND, Not detected. Seven contaminated sediments (Table I) were collected with a Petersen dredge from upper Escambia Bay and lower Escambia River at depths of 0.6 to 9 m. Beach sand and additional samples of sediment collected upstream from the contaminated section of the river served as uncontaminated controls. Four kilograms of each of the sediments and ten adult pink shrimp (7\ duararum) from a population which had previously shown no contamination with 'Aroclor' were placed in each of nine aquaria. Water at 17" C (lowing at a rale of 12 l./h, with an average salinity of 27 parts per thousand (p.p.t.), covered the substratum to a depth of 10 cm. Fiddler crabs (U. pitgilntor) were exposed to 'Aroclor' by placing them on live sediment substrates colloctcd as described. Four kilograms of each of the sediments were placed in tho Fiddler crabs and shrimp exposed to the contaminated sediments accumulated `Aroclor 1254' in their tissues by ingesting contaminated sediment particles or by absorbing (he leached chemical from water. They sifted the sediments before ingesting the particles. In most caves, the amount ol `Aroclor' in individual crabs or in shrimp hcpatopancreas "as directly refitted to the amount in the sediments. We analysed the distribution of the chemical in Ihc sediment in relation to particle size and found it was evenly distributed. Large con centrations of `Aroclor' residues may also accumulate m the hcpatopancreas of shrimp exposed to sandy silts because the chemical leaches from the sediments and is absorbed through the pills. Three and a half parts per billion (p.p.b.) were in the effluent water from the aquarium with sandy silt (61.0 p.p m `Aroclor'); the water from the silt (30.0 p.p.m.) had only 0.5 p.p.b. Even though fiddler crabs were not contintimi'-ly covered with water, they could have obtained `Aroclor- from the water as they wetted their gills. We have demonstrated experimentally that `Aroclor 1254 can cnier the estuarine food chain from sediments. Others4 have iilcntibi'd ihischcmie.il in higher trophic life such as itsh. and since I'CHs arc widespread in tlio wot IdJ, we should DSW 0 3 2 8 8 4 STLCOPCB4016846 Nwme more alert to lire presence, movement and cfleets of -ibrsc chemicals in iho environment. i D. R. Nimmo T. D. Wilson R. R. Dlackman A. J. Wilson, jun. VS Environmental Protection Agency, Gulf Breeze Ixi/mr.itory, - Guff Breeze, Florida 32561 -JleeeivcJ Soplcml'cr 17; revised Dcccmlwr 10, 1970. * Reynolds. L. M.. Hull. Emir. Contain, ami Toxicol., 4, 128 fl9(i9). * Rischiovph. R. w,, and Hrodme, V.. Environment, 12, 10 (1970). * Risehronch. R. W.. Kieelic. 1'.. 1'e.ik.ill. D. it.. Herman. S. G .aiul Kirven. M. N,, Nature, 220, ID'JS (196S): Koenian. J. H., Ten Noever Do llrauw, M. C.. ami Dc Vos, R. H., Nature, 221, 112(> (1909); Jensen, S., Jolmcls, A. G., Olsson, M., and Otlrrlind, G., Nature, 224, 247 (1969). * Duke, T. \V., Lowe, J. I., and Wilson, jun., A. J., Dull. Enriron. Contain, ami Toxicol., 5, 171 (1970). * Mill*. P. A., Onlcy, J. 11., and Gaither, R. A., J. Assoc. OJpe. Agrlc. Chon., 46, 186(1963). We propose the trivial name, 4-ipomcaqol, for this compound Which is a dihydro derivative of ipomeaninc, l-(.V-ftiryl)-|,4- pentanedione, .another known metabolite of sweet potatoes*. There arc nosrrports indicating toxicity for ipomeaninc. The empirical formula of d-ipomcanolrCnlluO, (molecular weight 168), was deduced from the mass spectrum by exact mass measurement of tire P-IljO ion. (We measured a value of 150.0700.002; C,ll|0Oj requires a value of 150.0GS.) The fl-furylkcto moiety was indicated by the characteristic nuclear magnetic resonance (NMR) signals for the furyl protons1, by the stretching frequency of the conjugated carbonyl group, by the ultraviolet spectrum* and by the intense mfe 95 ion ((J-C.HjO-CCM). The third oxygen function is a hydroxyl group (v 3,390 cm-1). Double irradiation NMR Showed that the hydroxyl group was embodied in a methyl Carbinol. Thcjjjcajnijgl shifts of the remaining two methylene groups were qfjwflsfcnt with the unbranchcd form of the structure. . A Lung Oedema Factor from Mouldy Sweet Potatoes (Ipomoca batatas) We have already described the toxic properties of sweet potato tubers infected with common moulds1 lJ. The toxic properties arc not directly attributable to the fungi because poisons were not found when the organisms were grown in . autoclaved sweet potato slurry. The experimental infection of viable sweet potato slices or of the intact tubers nevertheless . -resulted in the production of many furanoterpenoid com pounds, some of which arc markedly toxic for laboratory and commercial animals. As well as the previously recognized hepatotoxins ipomcaitiarone [(+ )-ngaionc]J and ipomcamaronol2, we have also isolated a more potent toxin called lung oedema factor (LO) from ether and chloroform extraeLs of both artificially and : -naturally contaminated sweet potatoes1, which recalls that natural outbreaks of mouldy sweet potato poisoning in cattle all dcscrilc respiratory distress related to lung oedema as the principal sign of disease*1*. On the other hand, hcpatotoxicily which might be attributable to ipomcamaronc or ipomca maronol has not been reported in most enzootics. Fresh tuber slices inoculated with sweet potato slurry-shake cultures of Fusarium javcnicum and incubated for approximately 6 days at 22-24u C contained significant quantities of all three toxins. We partially purified these metabolites by partitioning crude ether extracts of infected sweet potatoes on silica gel columns using elution with a hexane-ethyl acetate gradient. The column cluatcs were monitored by thin-layer chromato graphy (TLC) (silica gel; 9 ; 1 benzene-methanol). Ehrlich's Teagent produced twelve to fifteen coloured spots representing furanoterpenoids on TLC strips developed from the crude ether extract. Preliminary toxicity assays in mice showed that the LO factor was associated with a reddish-purple spot at Ai -- 0.4 which contained numerous compounds by gas chromatographic analysis. Column cluatc enriched with LO, consisting chiefly of the reddish-purple TLC spot material, was purified further by preparative gas-liquid chromatography (GC) of trimethylsilyljted (TMS) product. A single GC peak corresponding to the TMS ether of lung toxic material was obtained. We removed the TMS group by acid hydrolysis to give pure toxin, a colourless oil with a pungent odour. Spectral evidence (Table J) indicates that its structure is l-(3'-furyl)-4-hydroxy-l. pentanone.. Table 1 Spectra o! 4-lpomoanol Infrared, Ultraviolet cm"1 (neat) nm (cyclo hexane) 3,480 (OH) 243 (c 3,000) 3,110 (furyl) 211 (e 5,100) 2,940 2,900 1,675 (conj. C=0) 1,560 (furyl) 1.510 (furyl) 1,160 880 (furyl) NMR, 8 p.p.m. (CCL) MS. m/e(X) 1.15 (3H, d. J = 6Hz, 5-CHj) 1.67 (2H, m, 3-CHj) 2.50(111, broad s, OH) 2.83 (2H, t, J = 7Hz, 2-CMj) 3.73 (IH, m, 4-CH) 6.75 (1H, m, 4'-CH) 7.42 (IH. m, 5'-CH) 8.07 (IH, m, 2'-CH) 168 (5) Molccular ion 153 (2) M-CII, 150 (6) M-11,0 124 (5) M-CHjHCO 121 (2) M-H,0HCO 110 (47) OH fury1-i=CH, 95 (100) furyl-CO X ' , 4-Ipomcanof is acutely toxic to mice, producing the lung oedema and pleural effusion previously observed with crude sweet potato extracts. Doses of toxin (1-6 mg) given inlra- pcritoneally t$F;rnicc produced immediate signs of general illness, follow^titrickly by dyspnoea which gradually increased in severity to the dime of death, apparently from anoxia, in 5 to 8 h. Conjparablc quantities given orally were usually followed by a Short period of apparent latency (2 to 3 h) followed by increasing respiratory distress leading to death within 24 h. At post-mortem examinations, clear fluid, sometimes its much ns 1 ml. or more, dripped from the nostrils when the animal carcass was held with its head downwards. The lungs were frequently congested and surrounded by 1-2 ml. of clear pleural fluid. .Microscopic sections of the lungs stained with hasmatoxvlin and cosin showed intrn-alvcnlar oedema and congested blond vessels. In some animals there was a slight increase in p.fclnminal fluid but we observed no significant pathological changes in abdominal viscera. We have alsaisolatcd an isomer of 4-ipomc.mol, tentatively assigned the .structure 5-(3'-furyl)-5-hydroxy-2-pcntanonc, which is under.investigation. Sweet potatoes olfercd for sale in food stoics often contain ipomcamuroncv.jpomL\amaronol and 4-ipomcanol', The exact DSW 032885 STLCOPCB4016847