The Case of Diethylstilbestrol Treated Veal Contained in Homogenized Baby-Foods in Italy. Methodological and Toxicological Aspects

Ann. 1st Supw Sonitd Vol. 20. N 2-3 (1984),pp. 215.120

THE CASE OF DIETHYLSTILBESTROL TREATED VEAL CONTAINED IN HOMOGENIZED BABY-FOODS IN ITALY. METHODOLOGICAL AND TOXICOLOGICAL ASPECTS

t A. LOIZZO la), G.L. GATTI (b), A. MACRI' (C), G. MORETTI (d), E. ORTOLA'UI (a) and S. PALAZZESI (a)

(a) Laboratorio di Farrna~olo~ia;(6) Laboratorio di Tossicologia Applicata; (C) Laboratorio di Tossicologia Comparata ed Ecotussicolog.ia; fd) Labomtorio di Chimica del Farmaco, Istituto Superiore di Sanitd, Roma

Summary. - From previous studies carried out in the pericolose, di tali sostanze, con livelli dell'ordine di Istituto Superiore di Sanità, Romq over the years grandezza di 0,1-2 gg per kg di carne. E' stata quindi 1979-1980, it was hypothesized that calfmeat-derived, auanzata l'ipotesi che le elevate quantità di DES riscon- homogenized and lyophilized baby food might contain trate nei prodotti omogeneizzatiprouengano da carni di high doses of compounds endowed with oestrogenic ac- uitello nelle quali siano ancora presenti residui di impian- tiuity. Thus, anolytical inuestigations performed on ti (oppure di preparazioni iniettabili) di questa sostanza, 450samples of commercia1 baby-foodproducts, showed somministrata ai vitelli a scopi ouxinici Analoghe that 150 of them contained o powerful oestrogenic considerazioni sono state avanzate inoltre per spiegare la substance, Inter on identified as diethyistilbestrol (DES), eziopatogenesi delle epidemie di ginecomastiq pseu- in high amounu (20 to l40 @/kg food). Since meat do-pubertà precoce, oppure disturbi del2'apparato from calves illegally treated with xenobiotic drugs hauing sessuale di bambini in età scohre, descritti in Italia hormonal octivity generally contains modest yet poten- e in altripaesi del mondo in questi ultimianni tiolly dangcrous leuels ojsudi compounds (in the range 0.1 to 2 pglkg meot), it was derived that the eleuated DES levels in homogenizedproducts could be originated jiom working plout or injectable preparation DES residues, giuen to animais m ouxin Similar considerations Un April 1980 the Veterinary Se~cesof Italian ore advanced to exphin the etiopathogenesis of gynoe- Ministry of Health hecame aware of increasing trade comastia, early pseudopuberty, or troubles in the sex abuse of synthetic anabolic hormons as auxinic agents organs of school-age children described in Italy as well in animal farms [I]. as in other countries during these latter years. Moreover,a reportfrom paediatrichospitals on crypto- togenic hreast enlargement cases in prepuheral cliil- Riassunto (Il caso degli omogeneizzati contenenti dren [Z], induced Italian Health Authorities to con- carni di vitelli trattati con dietilstilhestrolo in Italia. sider the possibility tbat the use of hormons as aux- Aspetti metodologici e tossicologici). - Studi precedenti, inic agents in animal hreding was much larger than compiuti nell'lstituto Superiore di Sanità nel 1979-80, official information could suggest. Following indica- avevano suggerito l'ipotesi che alimenti omogeneizzati e tions contained in these studies [l],a nationwide pro- liofilizzati per bambini confezionati con carni di vitello, gam on hormons in calf meat nas devised in May potessero contenere forti dosi di sostanze ad attività 1980, and widespread controls vere therefore carried estrogena Infatti, una serie di indagini e determinarioni out by regional lahoratones on calf meat-containing analitiche, eseguite successivamente con metodi biologici children food. Over the period May-Jiily 1980, 450 e chimici su campioni di alimenti omogeneizzati preleuw sainples o€ baby food manifactured hy 5major producers ti dal commercio, ha evidenziato che 150 m 450 cam- in Italy (daily pruduction of about 300.000 jars) ran- pioni esaminati contenevano una potente sostanza dornly selected from 26 towns scattered al1 over Italy, esirogenq successivamente identificato come dietilstil- were examined. The meat was 37% of italian origin, bestrolo (DES), in dosi elevate (da circa 20 a 140pgper 25% imported from other EEC countries, 2.%from kg di prodotto omogeneizzato). Risulto tuttauiu che le european non EEC countries, 29%irorn South American carni commestibili dei uitelli, trattati illegnlmente con countries, 7Qofromotl~ers. 'l'lieee 450 saniples underwent sostanze xenobiotiche ad attiuità ormonale, contengono biologica1 assay for estrogenjc activity in regional contro1 in genere quantitàpiù modeste, anche se potenzialmente laboratories (see "Biolngical inetlioil"). Of these samples, 150 u.ere found to he positive for estrogenic acetic anhydride, heating and ohserving typical fluo- activity, therefore 82 out of these 150 werr forwarded rescence at 366 nm. As shown with added DES, the to our lahoratory for a second instance controì, while detection Iunit of this method was 2-3 pdkg food. A the remaining 68 were sent elsewherr. direct inlet mass specìrometry procedure was also used This report deals with the results ohtained from [7] in 2 out of the 10 samples. A pari of the extract pre- analyses performed on ttiese 82 samples. Potential ef- pared for TLC analysis (see ahove) was treated with fects induced hy auahohc-trcated meat on public health pentafluorobenzoyl chloride and triethylamine in ben- are also discussed. eene solution. Then, the reaction mixture was purified by silicic acid column chromatography. The part of eluate containing the pentafluorohenzoyl Methods derivative of DES was concentrated and transferred into the probe of tbe mass spectrometer (LKB 2091 in- a) Biologico1 method. - The method was hased on stmment). The prohe temperature was programmed up the weight increase of uteri in immature female mice, o- to 250 'C and the ionization energy was 70 eV. Mass riginaily set forth hy Umherger et aL [3] and Later mod- spectra were elahorated hy a computer system by ified hy Tiecco [4], as adopted hy the italian health repetitive scanning. authorities [5]. Groups of six 21 + 1 days old female mice were fed for 3 consecutive days with the sample to he analyzed, mingled with an equal quantity (wlw) of ground pellet food. On the 4th day the mice were sacrificed, and uterus weight (expressed as percent of Seventy-eight oi the eighty-two baby food samples hody weight) was compared with uterus weight 01 showed marked uterotropic activity. In Tahle 1 is de- groups of control mice, fed with the usual food, and of picted the distrihution in classes of mean uterus weight groups of mice fed estrogen free calf meat mingled with of mice treated with baby iood samples, expressed as an equai quantity (wlw) of ground pellet food. Where percent of the control, and the corresponding dose of estrogenic activity was found, an estimate of its amount DES, estimated on a previously estahliahed titration in terms of diethylstilbestrol (DES) equivalents was curve [8]. Ouly four out of 82 samples did not confirm made hy serial dilution of the sample with the control regional laboratories analysis at the l%significance level meat. and by comparing the respouse therehy ohtained (biologica1 method). The measurement of uterotropic with a standard dose/response curve aimuitan&usiy activity obtained hy dilution of 4 samples, moreover, ohtained hy addiug known amounts lof DES to the confirmed the afore mentioned estimation (Fig. 1). control meat diet. Moreover, since total amount of each sample (60 to 120 g per jar) did not always allow com- Tahle 1. - Distribution in chses of uterine weight in plete analysis (serial dilution sample plus chemical immature mice fed with baby food mingled 50% with anaiysis) semiquantitative measurement of uterotropic usual food, for 3 days activity was carried out also by dilution on 4 samples randomly chosen among those with marked uterotropic Utew weight increpse Number of Esfimited DES activity (range: 344 up to 503 percent of control). (in percent of control) smples content (in P&B fod b) Chemical method and ma-spectrometry. - The chemical method deseribed hy Verheke [6] for the from t0 deteetion of anabolic snhstances in animal tissues was employed on a total of 10 samples selected at random from the sampleswhich resulted positive by thebiological method described ahove. Baby food samples, 40-50 g, were extracted with methanol after enzymic hydrolysis with glucuronidase- sulphatase mixture in huffer solution. Tbe aqueous methanol phase was washed with n-hexane, and extracted with dichlorometane. The extract was evaporated to dryness. The residue was dissolved in water and al- lowed to percolate through an Amberlite XAI-2 column; the anabolic substances were eluted with methanol. The eluate was concentrated and then passed through a basic Extrelut column; estrogenic suhstances Resuits ue expreased percent of control. Four out of 82 were then eluted with diethyl ether from the column. samples did not confirm peripherd Iiboratories mdyeis at ths After concentration the extract was analysed hy two- 1% significame level. A nomd distribution of vdues ie evident. dimensioual thin layer chromatography using precoated In the ri&t pari of the tnble m esiimated DES content wss dica gel 60 HPTLC-plates. Estrogenic substances were drawn, eompanng the uterine weight with n titrstion cum detected hy spraying the plates with sulphuric acid in obtiuied with sevemi dosea of DES (for det& see ref. n. 8) EFFECTS INOUCEO BY OIFFERENT COIICENTRATIONS OF Diseussion ilOMOOENlZE0 MEATON UTEAINE WEIGHl IN MICE l The presence of a potent estrogenic substance was demonstrated in 78 out of 82 samples of commercial, homogenized haby food. Analytical investigations performed in 10 out of 78 samplea demonstrated that the estrogenic substance responsible for the utetua enlargement was DES and in two cases the presence of DES was confirmed by mass spectromehy. It is reason- able, therefore, to ipothesize that al1 other samples with marked uterotropic activity contained the same or eqnivalent compound. Overall, a figure of 33% of estrogen-positive jara among those anaìyzed al1 over the national temtory was found. If we postulate the presence in baby food of 20 to 140 &/kg of DES in 113 of baby food jam on sale in that period (see Fig. 1. - Titration curve obtdned through successive dilution of "Results"), we can estimate that children regularly 4 different baby food smples. Esch point repreaente meui of 24 fed with those baby food jars may have ingested 0.10 uiUnJs i stmdud deviation to 0.70 ~g DES per kg body weight per day, perhaps for severa1 months after weaning. In the last few years sporadic reports have been The chemical method used for 10 out of the 78 bio- published on cryptogenic epidemica of gynaecomastia, logically positive samples revealed the presence of or on cases of pseudopubertai proecox in children, DES: no other estrogenic suhstancea were detected. even aging 1 year [9,10]. In tbese reports, hypothesis A semiquantitative estimation of DES content in the of eatrogen contamined food had been forwarded, but samples was poseible by nsual comparison on dica gel no definite cause-effect relationship was demonstrated plates between sample and reference DES spots. These under controlled experimental conditions. estimations were in good agreement with the'evaluatiom According to the data ahown by Weber et oL [lo] performed with the biologicaì msay. Mass spectromehy doses of DES similar to the ones found in baby food, further confiied the identity of DES in the two al- are capable, upon chronic administration, to induce in ready biologically and chemically tested haby food sam- children various disturhances, chiefly consisting of ples. Figure 2A shows the mass apectnim ohtained for breast enlargement, vaginal bleeding and pseudopreco- the reference DES derivative, and Fig. 2B shows the cious puberty [Il]. However, symptoma descrihed in mms spectrum obtained from a sarnple positive by bio- older children by Paaquino et aL [Il], as well a0 in logica1 and chemical analyses; in this latter spectnim children described by Fara et al. (91, could not be DES is endenced a0 pentafluorobenzoate derivative induced by estrogens contained in baby food, which is (molecular ion [M]' 'mlz 656). aeldom adminietered to children after 18 months of age,

2.- W 60- L .-C

O ...... - 40- S 656 [M]*.

20 -

I.; , , , ,, , m.,,,, ._...... ,. .I.. .A,, 100 200 3M) 100 500 600 m/=

Fig. 2A. - Mzse apecirum of the pentafluorobenzoate derivate of reference diethylstihstrol Fig. 2B.-Mass speetnun of the pentafluorobenzoate derivative of diethyletilbestrol obtiined from a sample of baby food whieh resuited positive nt the biologica1 and ehemicd assays

therefore a different mechanism of ingestion of the mice or castrate male mice (25-500 pg DES/kg food) drng should be hypothesized. The illegal use of ana- with a latenq of 31 to 69 weeks [19]. ie. about 40 to bolic agents in cattle generally occurs through injec- 800'oof expected duration of life in a monse. tion or subcutaneous pellet implantation, of 30-72 mg Data collected from animal species have but indica- DES [12, 131. When implant site is discarded just be- tive values, and cannot be extrapolated on the human fore or after slanphtering, DES levels in meat or liver species. are usually low, up to a few pg DES per kg edihle Our data suggest that an nnexpectedly great numher tissue [13]. However, subcutaneously, or intramuscu- of children were probably exposed to high doses of larly, implanted DES is often not withdrawn, there- DES. The possible damage induced by DES on such chil- fore massive amounts of DES (10 to 20%of the original dren, cannot be easily established, but epidemia of amount of 30-72 mg) remain in the tissue, even for pseudoprecocious puberty have been descnbed both severa1 months, and are processed when the cattle is in Italy and abroad. Anabolic agents such as DES or being prepared for baby food, meat-balls, hamburgers, zeranol, present through the production of meat conld or as food stuff in genera1 (ie. 3 to 14 mg DES per be responsible for such episodes [2,8, 20-221. carcass, which is 150 to 200 kg). We would therefore underline that our data, as well These amounts are potentially high enough to induce as conclusions given in recent Intemational Symposium scattered episodes of homonal hyperdosage effects, as on anabolic hormones [23], strongly support conclusions prohably happened in some of the cases descnbed by [24] given by EEC Scientific Committee: ie. studies Fara et al. [9] and by Pasquino et aL [Il]. on acceptable daily intake values of xenobiotic hor- If the evaiuation of nsk inherent to the presence mones should conaider more carefully bioavailability, of DES in foodstuffs is considered, various aspects pharmacology and toxicology of minute amounts of of DES toxicity in animals may be quoted versus the these snbstances before accepting them as auxinic agents quantities of DES found in the course of food controls in animal farms 1251. or studies; minimai dose ranges (ie. DES levels between 0.1 and 2 pgikg food) are usually found in meat of implanted veais np to 30-40 days after treatment, and, conversely, in rata fed with this meat for 1 to 2 years histological and functional aiteration of repro- This work is dedicated to the memory of G.L. Gatti, ductive organs and fertility are induced [12-151. DES levels hetween 1 and 10 pg/kg food are often fonnd in The suthors Hish ta thmk L. Boniforli for miss apectrorneter liver or kidney of implanted veals, sometimes in plasma mdyses. Thnnks ue &o expresaed to B. Boscherini,G. De Felip, or meat [13, 16, 171; a diet containing such amonnt of F. Di Ruzzn, E. Pdiola, R. Sahii. V. Simo md C.A. Zzpponi DES induces weight increment of uterus and cornifica- for ohcussion uid suggestions, uid to P. Campagna for accurate tion of vaginal epithelium in prepuberal or castrated editing of the manucript. mice or rats, after 3 to 7 days [3, 12, 181. Higher DES levels induced linear 106 dose-response cuwes with Riceuuto il 2 luglio l984 significant slopes for neoplasm fomation in female Accettato il 22 dicembre 1984 REFERENCES

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