Hydroxylamine moiety of developmental toxicants is associated with early cell death: a structure-activity analysis.

BACKGROUND: Cellular debris, an indicator of cell death, appears in limb buds of gestational day 12 rabbit embryos 4 hr after either a subcutaneous injection of hydroxyurea to pregnant rabbits or an injection of hydroxyurea into the exocoelomic cavities of the embryos. This episode of early cell death appears to be central to the teratogenic action of hydroxyurea. Several chemicals that are structurally related to hydroxyurea, and that possess a terminal hydroxylamine moiety (-NHOH), also produce limb abnormalities. METHODS: To investigate whether the hydroxylamine moiety is responsible for early cell death and, therefore, is likely to be associated with teratogenesis, five structurally related hydroxylamine-bearing chemicals (hydroxylamine hydrochloride, N-methylhydroxylamine hydrochloride, hydroxyurea, acetohydroxamic acid, and hydroxyurethane) were administered at equimolar doses to rabbits either by subcutaneous (8.55 mmol/kg) or intracoelomic (2.66 micromol/embryo) injection on gestational day 12. Five additional chemicals, structurally similar to the hydroxylamine-bearing compounds, but possessing a terminal amino group (-NH(2)) (ammonium hydroxide, methylamine, urea, acetamide, and urethane), were tested at equimolar or higher doses by an identical protocol. In a subsequent experiment, the antioxidant propyl gallate (3.0 mmol/kg or 1.30 micromol/embryo) was co-administered with the hydroxylamine-bearing compounds to determine its effect on early cell death. Embryos were harvested 4 or 8 hr after treatment and analyzed by light microscopy. RESULTS: Cellular debris was obvious in forelimb buds from embryos treated with the hydroxylamine-bearing compounds; however, none of the amino compounds produced an early episode of embryonic cell death. In all cases, the antioxidant propyl gallate prevented or delayed the early episode of cell death observed after treatment with the hydroxylamine-bearing compounds. CONCLUSIONS: These results are consistent with the concept that the rapidly occurring embryonic cytotoxicity induced by hydroxylamine-bearing compounds involves a free radical mechanism that requires the presence of a terminal hydroxylamine group for initiation.

Protective effect of liposome encapsulation on paclitaxel developmental toxicity in the rat.

Paclitaxel is an anticancer drug that has demonstrated severe embryotoxicity in chicks. This embryotoxicity is reduced by liposome encapsulation of the drug. The current study was designed to evaluate the potential of liposome encapsulation for reducing paclitaxel embryotoxicity in rats. Wistar rats were treated with paclitaxel on day 8 of pregnancy (plug = day 0) at doses of 0.67, 2.0, or 10.0 mg/kg intravenously. The same doses of paclitaxel encapsulated in liposomes were administered intravenously to other groups of animals. Control animals were given blank liposomes. Free paclitaxel produced maternal and embryotoxicity at 10.0 mg/kg with three of seven dams dying and resorption of all embryos in surviving dams. Liposome encapsulation at 10.0 mg/kg was not associated with maternal death and there were live fetuses on evaluation at term, although litter size was reduced and malformations occurred in surviving fetuses. At 2.0 mg/kg free paclitaxel, fetal weight was decreased and resorptions increased. Liposome encapsulation at 2.0 mg/kg produced litter results similar to those obtained in control animals given empty liposomes. Malformations were prominent at 2.0 mg/kg free paclitaxel and at 10.0 mg/kg paclitaxel in liposomes and included exencephaly/anencephaly, ventral wall defects, facial clefts, anophthalmia, diaphragmatic hernia, and defects of the kidney, cardiovascular system, and tail. Liposome encapsulation appeared to shift the developmental response to paclitaxel such that 10 mg/kg encapsulated drug produced effects similar to 2.0 mg/kg free drug. These results may have implications for drug delivery of therapeutic agents used during human pregnancy.

Embryotoxicity of free and liposome-encapsulated taxol in the chick.

Taxol, an inhibitor of microtubule disaggregation, is used in the therapy of breast, ovarian, and other human malignancies. The toxicity of taxol administration is due in part to the polyoxyethylated castor oil (Cremaphor) vehicle in which it is administered; taxol embryotoxicity appears also to be partially attributable to vehicle toxicity. Liposome encapsulation is a novel vehicle for drug administration. The administration of taxol encapsulated in liposomes was evaluated in the chick embryo. Albumen injections of taxol doses up to 30 micrograms/egg were used to characterize dose-response curves for free and liposome-encapsulated taxol, compared to liposome-only and saline-injected control eggs. Sixty percent embryotoxicity (death or malformation) occurred with taxol doses of 1.5 micrograms/egg. A 20-fold higher dose was necessary to produce the same degree of toxicity with liposome-encapsulated taxol. Curve-fitting equations were used to estimate median effective doses (ED50s) and slope functions of the dose response curves. The ED50 for taxol was more than an order of magnitude lower than that for liposome-encapsulated taxol. Estimated slope functions for the two dosage forms of taxol were the same, suggesting similar mechanisms of toxicity. The toxicity of liposomes alone was low.

D-mannitol, a specific hydroxyl free radical scavenger, reduces the developmental toxicity of hydroxyurea in rabbits.

Hydroxyurea (HU) is a potent mammalian teratogen. Within 2-4 hours after maternal injection, HU causes 1) a rapid episode of embryonic cell death and 2) profound inhibition of embryonic DNA synthesis. A variety of antioxidants delays the onset of embryonic cell death and reduces the incidence of birth defects. Antioxidants do not block the inhibition of DNA synthesis, indicating that early embryonic cell death is not caused by inhibited DNA synthesis. We have suggested that some HU molecules may react within the embryo to produce H2O2 and subsequent free radicals, including the very reactive hydroxyl free radical. The free radicals could cause the early cell death; antioxidants are believed to terminate the aberrant free radical reactions resulting in lessened developmental toxicity. To investigate whether hydroxyl free radicals cause the early episode of cell death, pregnant New Zealand white rabbits were injected subcutaneously on gestational day 12 with a teratogenic dose of HU (650 mg/kg) in the presence or absence of 550 mg/kg of D-mannitol (Man), a specific scavenger of hydroxyl free radicals. Osmotic control rabbits received HU plus 550 mg/kg of xylose (Xyl, a nonactive aldose). At term, the teratologic effects of HU were ameliorated by Man as evidenced by decreased incidences of the expected limb malformations. Xyl exerted no demonstrable effect on HU teratogenesis. Histological examination of limb buds at 3-8 hours after maternal injection, showed that Man delayed the onset of HU-induced cell death by as much as 4 hours. Xyl had no effect. That Man acts within the embryo was shown by performing intracoelomic injections on alternate implantation sites with Man, Xyl, or saline followed by subcutaneous injection of the pregnant doe with HU. Embryos were harvested 3-8 hours later. Limb buds from saline- and Xyl-injected embryos exhibited the typical pattern of widespread HU-induced cell death at 3-4 hours, whereas Man-injected embryos did not exhibit cell death until 5-8 hours. These results are consistent with those reported for antioxidant-mediated amelioration of HU-induced developmental toxicity and with the hypothesis that hydroxyl free radicals are the proximate reactive species in HU-induced early embryonic cell death.

Taxol and embryonic development in the chick.

Taxol, an inhibitor of microtubule disassembly, is currently under investigation in the therapy of several human cancers. The current investigation was undertaken to characterize potential taxol developmental toxicity in chicks. On one of days 1-4 of incubation, taxol was administered in dimethylsulfoxide (DMSO) or olive oil in a range of doses, the highest of which produced a high incidence of early embryo death. Production of gross structural malformations was sporadic and occurred in vehicle-treated as well as taxol-treated embryos. A more common manifestation of taxol toxicity was a syndrome of visceral abnormalities, including regression of the vitelline circulation, dilatation of the atria, and hemorrhage in the left side of the head and thorax, often with decreased eye pigmentation. Regardless of the day of treatment, this syndrome occurred at 4.5-5 days. To investigate the possibility that taxol induced its effect through disruption of angiogenesis in the vitelline circulation, filters soaked in taxol were applied to the margin of the germ disc. No inhibition of vessel development was demonstrated. We conclude that taxol decreases the viability of embryos and that this impairment of survival precludes the development of birth defects. Solvent toxicity is an important confounder in the investigation of taxol embryotoxicity.

Methotrexate-induced developmental toxicity in rabbits is ameliorated by 1-(p-tosyl)-3,4,4-trimethylimidazolidine, a functional analog for tetrahydrofolate-mediated one-carbon transfer.

Dihydrofolate reductase reduces folic acid to tetrahydrofolate as a prerequisite to one-carbon metabolism, which is required for normal embryonic de novo DNA synthesis. The developmental toxicity of methotrexate (MTX) has been attributed to MTX's ability to inhibit the activity of dihydrofolate reductase and thereby indirectly suppress one-carbon metabolism. The compound 1-(p-tosyl)-3,4,4-trimethylimidazolidine (TTI), which is structurally unrelated to folate, reestablishes one-carbon metabolism by the biomimetic transfer of single carbon units. Whether the developmental toxicity of MTX is indeed caused via suppressed one-carbon metabolism was tested in New Zealand white rabbits following concurrent maternal treatment with MTX and TTI. TTI reduced MTX developmental toxicity judged by increased mean fetal body weights, decreased percentage of malformed fetuses, and reduced incidences of major malformations. Two doses of TTI (90 mg/kg, each) at 1 hr prior to and 1 hr after MTX also reduced the developmental toxicity, but was no more effective than the single-injection regimen. Treatment with TTI alone caused no developmental toxicity. Histologically, MTX caused enlarged intercellular spaces in limb bud mesenchyme that began at 6-8 hr and increased in size until 16 hr. Mesenchymal nuclei appeared basophilic, with angular contours. Pretreatment with TTI delayed MTX-induced histological changes until 20-24 hr after MTX in 36-50% of embryos and completely protected the remainder. The sequence of MTX-induced changes was not altered among affected embryos, although the severity of the lesions did not appear as great. Saline-only or TTI-only treatments caused no alterations in limb buds. These data are consistent with the concept that impaired one-carbon metabolism is indeed the fundamental process underlying MTX developmental toxicity.

Amelioration by leucovorin of methotrexate developmental toxicity in rabbits.

Methotrexate (MTX) is lethal or teratogenic to embryos of all species tested. New Zealand white rabbit embryos are relatively resistant to the embryolethal effects of MTX. However, when pregnant does were injected iv with 19.2 mg MTX/kg on gestational day 12, virtually all surviving fetuses exhibited multiple malformations of the head, limbs, and trunk. MTX is a structural analogue of folic acid that competitively inhibits dihydrofolate reductase, thereby preventing formation of folinic acid and essentially stopping one carbon metabolism. One carbon metabolism is important in the synthesis of methionine, histidine, glycine, and purine bases that are required for the de novo synthesis of DNA. Presumably these metabolic effects of MTX relate directly to its mechanism of developmental toxicity. An ameliorative treatment has been tested utilizing i.v. injection of pregnant rabbits with leucovorin (LV), a close structural analogue of folinic acid (the product of the inhibited enzyme), at various times after MTX exposure. When LV was injected at times up to 24 hours after MTX fewer malformed fetuses resulted and the incidence of specific malformations was reduced. When given at times up to 20 hours after MTX administration, LV virtually eliminated the grossly apparent effects of MTX at term. In the forelimb bud, MTX increased the extracellular space surrounding limb bud mesenchymal cells within 8-10 hours; this process continued through 16 hours and remained unabated by 24 hours. Mesenchymal cell nuclei became hyperchromatic and pyknotic during this time period. By 24 hours, a moderate amount of cellular debris was observed in the mesenchymal compartment of limb buds from approximately one-third of the embryos examined. Endothelial cell nuclei of the limb bud vasculature did not exhibit the histopathological alterations observed in the mesenchymal cells. Limb buds from embryos injected with LV at times up to 6 hours after MTX were histologically normal. When LV treatment was delayed until 16 or 20 hours after MTX, mesenchymal nuclei regained normal appearance within 2 hours of treatment; further, the abnormally large intracellular space began to decrease during the next 4 hours. Cellular debris was not a prominent feature of limb buds from LV-treated embryos examined at any time. Embryos from rabbits injected with LV at 24 hours after MTX exhibited either typical MTX-induced lesions or a sequence of reparative events similar to those described for the 16 and 20 hour LV-treated embryos.(ABSTRACT TRUNCATED AT 400 WORDS)

Perfluorocarbon-based medium for culture of the early chick embryo heart.

In an attempt to prolong the survival of the explanted early chick embryo heart, hearts at stages 10 to 28 were cultured in supplemented Dulbecco's modified Eagle's medium with or without the perfluorocarbon, perfluorotributylamine. The perfluorocarbon was added to the standard culture medium in a 50:50 (vol/vol) mixture. Explants were evaluated daily and were harvested for light microscopy after 2 to 10 d in culture. The tubular shape of the explants was generally maintained for 2 d in culture, after which the hearts became dilated or spherical. Beating was noted in some of the explants on Day 2 in culture but not thereafter. Microscopic evaluation showed patchy areas of necrosis in all explants by Day 3, although large areas of viable epithelioid cells were documented as long as 7 d after explanation. Stage 16 to 18 hearts cultured in the presence of perfluorocarbon were more likely to maintain tubular architecture on microscopy than hearts cultured in standard medium. Hearts cultured from later stages showed no improvement in appearance with the presence of perfluorocarbon and there was a suggestion of increased necrosis in later-stage explants cultured with perfluorocarbon for 4 d. Further modification of the culture system will be required to prolong explant survival and development beyond 2 d.

Developmental toxicity of hydroxylamine: an example of a maternally mediated effect.

Hydroxylamine (HA) is an important reducing agent that is used in several industries. HA is a moderate irritant and a powerful inducer of methemoglobinemia. HA has been shown to react readily with DNA in vitro. Several chemical derivatives of HA are potent developmental toxicants, whereas HA has been reported to cause no developmentally toxic effects. Since HA and its developmentally toxic derivatives share the presence of a terminal hydroxylamine functional group (-NHOH), and since that functional group has been proposed to be the biologically active portion of the molecule, it was deemed appropriate to re-examine the possible developmental toxicity of HA. Subcutaneous or intravenous injection of pregnant rabbits with 50-650 mg HA.HCl/kg on gestational day 12 caused the death or sacrifice of all rabbits within 30 hours. All maternally injected rabbits exhibited severe cyanosis, presumably due to methemoglobinemia. Histological examination of embryos revealed alterations of the cardiovascular system at 5 hours, but an absence of cell death in limb buds. At 8 hours, all embryos were dead. These effects appear to be secondary to the observed cyanosis in the maternal animals. In order to circumvent the powerful methemoglobinemia-inducing properties of HA, intracoelomic injections of 25-200 micrograms HA.HCl in 5-40 microliters of saline were made directly into the chorionic cavity of developing rabbit embryos, thereby bypassing the maternal system. Controls received similar volumes of saline. At doses of greater than or equal to 75 micrograms, HA.HCl killed 91 percent of injected embryos; among survivors, all exhibited reduced birth weights and 2/5 exhibited malformations in the craniofacial region and sternum. At doses of 25-50 micrograms, HA.HCl caused increased resorptions compared with controls; however, surviving fetuses displayed neither malformations nor reduced birth weights. Histological analysis at 4 hours after injection revealed cellular debris in the limb buds; when the antioxidant propyl gallate was co-administered with HA, cellular debris was absent at 4 hours. It is concluded that although HA is a directly acting developmental toxicant if it reaches the embryo, the observed embryolethality seen after subcutaneous injection of pregnant animals is a consequence of the powerful maternal toxicity of HA.

The nature of the embryo-protective interaction of propyl gallate with hydroxyurea.

Hydroxyurea (HU) is a swiftly acting cytotoxic teratogen and an inhibitor of DNA synthesis. Within 2 h of maternal treatment, HU causes necrosis in proliferating tissues of rabbit embryos on gestational day 12. Co-administration of the antioxidant propyl gallate (PG) delays the onset of necrosis until 6 h and ameliorates the teratogenic effects seen at term. Since HU also causes a rapid, profound decrease in uterine blood flow in pregnant rabbits, it is necessary to determine whether HU and PG interact within the pregnant female or within the embryo. In order to establish that the site of HU-PG interaction is embryonic, HU, PG, HU-PG, or vehicle was injected directly into implantation sites. When embryos were examined microscopically at 4 h, necrosis was observed only in the HU-treated embryos, indicating that the palliative interaction between HU and PG takes place within the embryo. To resolve whether the alleviation of HU-induced embryotoxicity was due to decreased HU levels within HU-PG embryos, HU concentrations were measured in embryos from HU-and HU-PG-treated maternal rabbits at 15 min to 8 h post injection. The HU levels of the two groups differed significantly only at 4 h. The rates of uptake during the linear phase (times from 15 min to 3 h) did not differ. When HU concentration was plotted versus time, measurements of the areas under the curve also did not differ. To determine whether PG alters the HU-induced inhibition of DNA synthesis, 3H-thymidine incorporation into embryonic DNA was assayed at 2 h after HU, HU-PG, or vehicle injections.(ABSTRACT TRUNCATED AT 250 WORDS)

Ethoxyquin and nordihydroguaiaretic acid reduce hydroxyurea developmental toxicity.

Hydroxyurea (HU) is a potent teratogen that causes a characteristic, rapidly occurring episode of embryonic cell death 2 to 4 h after subcutaneously injecting 650 mg/kg of HU into pregnant New Zealand White rabbits on gestational day 12. Previous studies documented the ability of the phenolic antioxidant, propyl gallate, to delay the onset of embryonic cell death and to decrease the number and severity of defects seen at term. The present study investigated the ability of the structurally different antioxidants, ethoxyquin (ETX) and nordihydroguaiaretic acid (NDGA), to also ameliorate HU developmental toxicity. Injection of pregnant rabbits with either ETX or NDGA at 950 mg/kg, 15 to 30 min prior to HU injection, resulted in reduced developmental toxicity seen at term. The reduction was manifested by fewer malformed fetuses with increased body weights compared with fetuses from HU-only treated litters, greatly reduced incidences of specific malformations, and diminished severity of some HU-induced defects. Microscopic analysis of HU, HU-ETX, and HU-NDGA embryos was performed at 4 or 8 h after treatment. HU caused cell death in the limb bud mesenchyme, clearly evident at 4 h. In contrast, HU-NDGA embryos exhibited no signs of cell death until 8 h after treatment. Although most HU-ETX embryos exhibited little or no cell death at 4 h after treatment, in about 20% the level of cell death was indistinguishable from that in HU-treated embryos. The amelioration of HU developmental toxicity in the present study is consistent with the results of previous studies utilizing propyl gallate. The results suggest that the antioxidant properties of these substances interfere with the rapidly occurring toxic effects of HU and that this may account for amelioration of HU developmental toxicity.

Lectin teratogenesis. II: Demonstration of increased binding of concanavalin A to limb buds of rabbit embryos during the teratogenically sensitive period.

The plant lectin concanavalin A (con A) causes malformations of rabbit embryos when 160 micrograms (in 40 microliter) are injected into the exocoelom on gestational days 12-15 but does not cause malformations on days 10-11. The purpose of this study was to investigate the mechanism for increased susceptibility of day 12-15 embryos to con A teratogenicity. Light microscopy of day 11 embryos 15-20 hr after treatment with con A revealed no observable difference from controls. Day 13 embryos at similar times exhibited limb buds with large areas that were denuded of ectoderm. Concurrent addition of alpha-methyl-D-mannoside (alpha MM), a specific inhibitor of con A, to the injection solution of day 13 embryos resulted in limb buds that appeared normal. The regions of con A binding to day 11 and day 13 embryos were visualized through epifluorescent microscopy of untreated embryos stained with fluorescein-labelled con A. Day 11 embryos exhibited moderate fluorescence on the surface of limb buds and the pericardial region. Day 13 embryos exhibited strong fluorescence of limb bud surfaces; the pericardial region remained moderately fluorescent. Addition of alpha MM to the incubation medium resulted in no fluorescence above background. Visualization of con A receptors was accomplished by ultrastructural analysis of forelimb buds stained with ferritin-labelled con A. Ferritin label was observed only on the surfaces of the ectoderm and was sparse over all regions of day 11 limb buds. In contrast, ferritin label was moderately heavy in all regions of the day 13 limb buds. No labelling occurred when the ferritin-labelled con A was preincubated with alpha MM. These observations indicate that the number of exposed con A receptors on limb buds of teratogenically sensitive embryos (day 13) is increased, compared with the number of exposed receptors on limb buds of younger, insensitive (day 11) embryos. The increased number of exposed con A receptors on limb buds during the teratogenically sensitive period provides not only increased binding of the lectin to sensitive embryos but also a potential mechanism for the anomalous attachment of distal regions of the limb buds to the body wall.

The embryogenesis and anatomy of Waldeyer's ring.

The developmental history of the embryonic germ disk is briefly traced through the bilaminar, trilaminar, and bodyfold stages with emphasis on the pharyngeal arches, pouches, and clefts. This is followed by a discussion of the genesis and adult anatomy of the tonsillar tissues.

Pancreatic elastase activation as a possible indicator of the relative hazard of different cigarettes.

Levels of elastase activity in homogenates of pancreas from beagle dogs exposed to cigarette smoke on a daily basis for 600 d were previously reported. Elastase activity in pancreatic homogenates from animals exposed to smoke from high-nicotine cigarettes was significantly greater than in sham-exposed controls or in animals smoking low-nicotine cigarettes. In the study described here, dogs were exposed on an acute basis to smoke from high-nicotine and from nicotine-free cigarettes. Anesthetized animals were prepared by laparotomy, duodenotomy, and pancreatic duct cannulation. Pancreatic fluid was collected and preexposure elastase levels were determined. Samples were then taken during exposure to smoke from cigarettes of both types. There was no measurable elastase activity in the baseline samples. Similarly, no activity was found in fluids collected before and during exposure to smoke from nicotine-free cigarettes. Enzyme activity was detected, however, in samples collected during exposure to smoke from high-nicotine cigarettes once blood levels of nicotine reached 50-70 ng/ml. The results suggest that cigarette smoke may provoke activation of elastase and indicate the need for further study of any association of cigarette smoking with pancreatic disease.

Lung development in the chick embryo. I. Phosphatidylcholine synthesis in the developing chick lung.

We investigated the relative contribution of the two pathways of phosphatidylcholine (PC) synthesis in lungs from chick embryos of 12 to 20 days of incubation as well as from hatched chicks and adult chickens. Lung "blocks" were incubated for 60 min in vitro in the presence of [3H]methionine and [14C]choline. PC was separated by thin-layer chromatography, and the amount of labeled product was determined by liquid scintillation counting. PC synthesis (expressed as pmoles PC per mg protein per 60 min) by the choline pathway declined from the 12-day value of 40 to a minimum of 8 at day 16 and then rose gradually to a peak of 74 on day 1 posthatching. PC synthesis via the phosphatidylethanolamine pathway also declined from day 12 (54) to day 16 (13) but peaked on day 19 (65) and again on day 1 posthatching (54). Synthetic activities of the two pathways differed significantly (P less than or equal to 0.05) only on days 14, 18, and 19 of incubation. Unlike the mammalian lung, neither pathway seems to predominate throughout incubation or after hatching. The increase in PC synthesis towards the end of incubation correlates with the time of appearance of lamellar bodies in the lung epithelial cells and of surfactant in the airways.

Lifestyle factors and cancer of the pancreas: a hypothetical mechanism.

The interaction of a genetically determined protease inhibitor, the enzymes whose functions are modified by that inhibitor and lifestyle factors, such as cigarette smoking, high lipid diet and alcohol consumption, are considered key factors in a proposed protease-antiprotease imbalance mechanism for pancreatic oncogenesis. Epidemiologic and experimental laboratory evidence in support of the mechanism is presented along with a discussion of suggested research initiatives to further test the hypothesis.

Pancreatic elastase and serum alpha 1-antitrypsin levels in beagle dogs smoking high- and low-nicotine cigarettes: possible mechanism of pancreatic cancer in cigarette smokers.

Beagle dogs exposed to cigarette smoke for 600 d experience a significant change in pancreatic elastase levels, as measured in tissue homogenates, compared with their sham-exposed controls. Greater elastase activity was found in the high-nicotine cigarette smokers than in the low-nicotine cigarette smokers. Levels of serum alpha 1-antitrypsin, an antiprotease capable of complexing the excess elastase, were also investigated. Animals smoking high-nicotine cigarettes had significantly lower serum alpha 1-antitrypsin activities than controls. Low-nicotine smokers showed alpha 1-antitrypsin activities that were not significantly different from those of controls. The importance of these observations is reinforced by a number of studies suggesting that proteases, their inhibitors, and an imbalance thereof may be related to the onset of neoplastic lesions. Studies have indicated that antiprotease levels follow the patterns of Mendelian inheritance. Severe deficiency states predispose human subjects to emphysema. A similar relationship may exist between antiprotease levels and susceptibility or resistance to neoplasms of the pancreas, a concept that deserves investigation in light of the findings reported here.