Impact of Food Matrices on Digestibility of Allergens and Poorly Allergenic Homologs.

Background: Protease resistance is considered a risk factor for allergenicity of proteins, although the correlation is low. It is nonetheless a part of the weight-of-evidence approach, proposed by Codex, for assessing the allergenicity risk of novel food proteins. Susceptibility of proteins to pepsin is commonly tested with purified protein in solution. Objective: Food proteins are rarely consumed in purified form. Our aim was to evaluate the impact of experimental and endogenous food matrices on protease susceptibility of homologous protein pairs with different degrees of allergenicity. Methods: Porcine and shrimp tropomyosin (ST) were subjected to sequential exposure to amylase, pepsin, and pancreatin in their respective endogenous matrix (pork tenderloin/boiled shrimp) and in three different experimental matrices (dessert mousse [DM], soy milk [SM], and chocolate bar [CB]). Digestion was monitored by immunoblotting using tropomyosin-specific antibodies. Recombinant peach and strawberry lipid transfer protein were biotinylated, spiked into both peach and strawberry fruit pulp, and subjected to the same sequential digestion protocol. Digestion was monitored by immunoblotting using streptavidin for detection. Results: Chocolate bar, and to a lesser extent SM, had a clear protective effect against pepsin digestion of porcine tropomyosin (PT) and to a lesser extent of ST. Increased resistance was associated with increased protein content. Spiking experiments with bovine serum albumin (BSA) confirmed the protective effect of a protein-rich matrix. The two tropomyosins were both highly resistant to pepsin in their protein-rich and lean native food matrix. Pancreatin digestion remained rapid and complete, independent of the matrix. The fat-rich environment did not transfer protection against pepsin digestion. Spiking of recombinant peach and strawberry lipid transfer proteins into peach and strawberry pulp did not reveal any differential protective effect that could explain differences in allergenicity of both fruits. Conclusions: Protein-rich food matrices delay pepsin digestion by saturating the protease. This effect is most apparent for proteins that are highly pepsin susceptible in solution. The inclusion of food matrices does not help in understanding why some proteins are strong primary sensitizers while homologs are very poor allergens. Although for induction of symptoms in food allergic patients (elicitation), a protein-rich food matrix that may contribute to increased risk, our results indicate that the inclusion of food matrices in the weight-of-evidence approach for estimating the potential risks of novel proteins to become allergens (sensitization), is most likely of very limited value.

Review of animal models designed to predict the potential allergenicity of novel proteins in genetically modified crops.

The safety assessment of genetically modified crops involves the evaluation of the potential allergenicity of novel proteins by using several in silico and in vitro endpoints. In this publication, the variables and questions associated with the development of in vivo models are examined and several unpublished results are presented. Both rodent and non-rodent (dog and pig) models have been investigated using various routes of administration with purified proteins or food extracts, with or without the use of an adjuvant. The ideal model should be simple, reproducible across laboratories over time, specific and sensitive enough for distinguishing a threshold beyond which relevant allergenicity would be predicted and, for ranking proteins correlated with the allergic responses in humans, and acceptable under animal care. Preliminary data suggest that a few appear promising; however, further evaluation of these models is required. In particular, more extensive validation testing with additional allergenic and non-allergenic material should be performed before using them in the safety assessment of genetically modified crops.

In vitro digestibility of beta-casein and beta-lactoglobulin under simulated human gastric and duodenal conditions: a multi-laboratory evaluation.

Initially the resistance to digestion of two cow's milk allergens, beta-casein, and beta-lactoglobulin (beta-Lg), was compared using a "high-protease assay" and a "low-protease assay" in a single laboratory. The low-protease assay represents an alternative standardised protocol mimicking conditions found in the gastrointestinal tract. For the high-protease assay, both proteins were incubated with either pepsin or pancreatin and digestion monitored by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reverse phase-high performance liquid chromatography. The low-protease assay involved gastroduodenal digestion in the presence or absence of phosphatidylcholine (PC). Both beta-casein and beta-Lg were susceptible to hydrolysis by pepsin and pancreatin in the high-protease assay. In contrast, the kinetics of beta-casein digestion in the low-protease assay were slower, beta-Lg being pepsin resistant. During duodenal digestion, beta-Lg was gradually degraded and addition of PC slowed digestion. Subsequently, the reproducibility of the low-protease assay was assessed in 12 independent laboratories by visual assessment of the gels and densitometric analysis: the inter- and intra-laboratory variability was affected by sampling and electrophoresis method employed. The low-protease assay was shown to be reproducible. Future studies will extend these findings using a broader panel of proteins.

Safety assessment of a modified acetolactate synthase protein (GM-HRA) used as a selectable marker in genetically modified soybeans.

Acetolactate synthase (ALS) enzymes have been isolated from numerous organisms including soybeans (Glycine max; GM-ALS) and catalyze the first common step in biosynthesis of branched chain amino acids. Expression of an ALS protein (GM-HRA) with two amino acid changes relative to native GM-ALS protein in genetically modified soybeans confers tolerance to herbicidal active ingredients and can be used as a selectable transformation marker. The safety assessment of the GM-HRA protein is discussed. Bioinformatics comparison of the amino acid sequence did not identify similarities to known allergenic or toxic proteins. In vitro studies demonstrated rapid degradation in simulated gastric fluid (<30s) and intestinal fluid (<1min). The enzymatic activity was completely inactivated at 50 degrees C for 15 min demonstrating heat lability. The protein expressed in planta is not glycosylated and genetically modified soybeans expressing the GM-HRA protein produced similar protein/allergen profiles as its non-transgenic parental isoline. No adverse effects were observed in mice following acute oral exposure at a dose of at least 436 mg/kg of body weight or in a 28-day repeated dose dietary toxicity study at doses up to 1247 mg/kg of body weight/day. The results demonstrate GM-HRA protein safety when used in agricultural biotechnology.

Current codex guidelines for assessment of potential protein allergenicity.

A rigorous safety assessment process exists for GM crops. It includes evaluation of the introduced protein as well as the crop containing such protein with the goal of demonstrating the GM crop is "as-safe-as" non-transgenic crops in the food supply. One of the major issues for GM crops is the assessment of the expressed protein for allergenic potential. Currently, no single factor is recognized as an identifier for protein allergenicity. Therefore, a weight-of-evidence approach, which takes into account a variety of factors and approaches for an overall assessment of allergenic potential, is conducted [Codex Alimentarious Commission, 2003. Alinorm 03/34: Joint FAO/WHO Food Standard Programme, Codex Alimentarious Commission, Twenty-Fifth Session, Rome, Italy, 30 June-5 July, 2003. Appendix III, Guideline for the conduct of food safety assessment of foods derived from recombinant-DNA plants, and Appendix IV, Annex on the assessment of possible allergenicity, pp. 47-60]. This assessment is based on what is known about allergens, including the history of exposure and safety of the gene(s) source; protein structure (e.g., amino acid sequence identity to human allergens); stability to pepsin digestion in vitro [Thomas, K. et al., 2004. A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins. Regul. Toxicol. Pharmacol. 39, 87-98]; an estimate of exposure of the novel protein(s) to the gastrointestinal tract where absorption occurs (e.g., protein abundance in the crop, processing effects); and when appropriate, specific IgE binding studies or skin prick testing. Additional approaches may be considered (e.g., animal models; targeted sera screening) as the science evolves; however, such approaches have not been thoroughly evaluated or validated for predicting protein allergenicity.

Assessment of three human FcepsilonRI-transfected RBL cell-lines for identifying IgE induced degranulation utilizing peanut-allergic patient sera and peanut protein extract.

Specific IgE sera screening studies are employed to investigate protein cross-reactivity. Such nonfunctional immunochemical methods cannot measure the biological activity of proteins. Therefore, an assay using RBL cells transfected with human FcepsilonRI was developed. Our objective was to evaluate the degranulation of three cell-lines expressing either the alpha-(RBL-hEI(a)-2B12 and RBL-30/25cells) or alpha-, beta-, and gamma-subunits (RBL SX-38) of the human FcepsilonRI by beta-hexosaminidase release. Purified human IgE and serum-derived polyclonal IgE from peanut-allergic subjects following challenge with anti-IgE or peanut protein extract, respectively, were utilized. Robust degranulation was induced in all three: RBL-30/25 (84%), -hEI(a)-2B12 (54%), SX-38 (94%), respectively, using purified IgE+anti-human IgE. Good release (18%, 40-45%, and 65%, respectively) occurred for one peanut-allergic subject+peanut extract with all cell-lines. With serum from three other peanut-allergic subjects, no beta-hexosaminidase release occurred with RBL-hEI(a)-2B12 cells+peanut extract, while only serum from one subject induced good degranulation, 30% and 60%, respectively, with RBL-30/25 and RBL SX 38 cells. Consistent degranulation with a potent food allergen (peanuts) was not observed. The assay's utility in safety assessment, predictive value and reproducibility for evaluating the cross-reactivity of proteins with allergens needs further investigation with additional proteins and well-characterized sera.

Cytokine mRNA profiles for isocyanates with known and unknown potential to induce respiratory sensitization.

Isocyanates are low-molecular-weight chemicals implicated in allergic asthmatic-type reactions. Identification of chemicals likely to cause asthma is difficult due to the lack of a validated test method. One hypothesis is that differential cytokine induction (Th1 versus Th2 profiles) in the draining lymph node following dermal application can be used to identify asthmagens and distinguish them from contact allergens. In this study, we compared the cytokine mRNA profiles of six chemicals: toluene diisocyanate (TDI), diphenylmethane-4,4'-diisocyanate (MDI), dicyclohexylmethane-4,4'-diisocyanate (HMDI), isophorone diisocyanate (IPDI), p-tolyl(mono)isocyanate (TMI), and meta-tetramethylene xylene diisocyanate (TMXDI). Whereas TDI and MDI are well-known respiratory sensitizers, documentation for HMDI, IPDI, TMI, and TMXDI is limited, but suggests that HMDI and IPDI may have respiratory sensitization potential in humans and TMI and TMXDI do not. Following dermal exposure of BALB/c mice, all six isocyanates induced cytokines characteristic of a Th2 response. Although LLNAs suggested that the doses chosen for the RPA were immunologically equivalent, the isocyanates tested differentiated into two groups, high responders and low responders. However, two of the low responders (TMI and TMXDI) were further tested and induced higher levels of Th2 cytokine message than dinitrochlorobenzene (not an asthmagen). Further study of these chemicals is needed to determine whether the Th2 cytokine responses observed for these low responders is predictive of asthmagenic potential or represents an insufficient signal.

A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins.

Rationale. Evaluation of the potential allergenicity of proteins derived from genetically modified foods has involved a weight of evidence approach that incorporates an evaluation of protein digestibility in pepsin. Currently, there is no standardized protocol to assess the digestibility of proteins using simulated gastric fluid. Potential variations in assay parameters include: pH, pepsin purity, pepsin to target protein ratio, target protein purity, and method of detection. The objective was to assess the digestibility of a common set of proteins in nine independent laboratories to determine the reproducibility of the assay when performed using a common protocol. Methods. A single lot of each test protein and pepsin was obtained and distributed to each laboratory. The test proteins consisted of Ara h 2 (a peanut conglutin-like protein), beta-lactoglobulin, bovine serum albumin, concanavalin A, horseradish peroxidase, ovalbumin, ovomucoid, phosphinothricin acetyltransferase, ribulose diphosphate carboxylase, and soybean trypsin inhibitor. A ratio of 10U of pepsin activity/microg test protein was selected for all tests (3:1 pepsin to protein, w:w). Digestions were performed at pH 1.2 and 2.0, with sampling at 0.5, 2, 5, 10, 20, 30, and 60min. Protein digestibility was assessed from stained gels following SDS-PAGE of digestion samples and controls. Results. Results were relatively consistent across laboratories for the full-length proteins. The identification of proteolytic fragments was less consistent, being affected by different fixation and staining methods. Overall, assay pH did not influence the time to disappearance of the full-length protein or protein fragments, however, results across laboratories were more consistent at pH 1.2 (91% agreement) than pH 2.0 (77%). Conclusions. These data demonstrate that this common protocol for evaluating the in vitro digestibility of proteins is reproducible and yields consistent results when performed using the same proteins at different laboratories.

Identifying airway sensitizers: cytokine mRNA profiles induced by various anhydrides.

Exposure to low molecular weight (LMW) chemicals in the workplace has been linked to a variety of respiratory effects. Within the LMW chemicals, one of the major classes involved in these effects are the acid anhydrides. The immunological basis of respiratory hypersensitivity involves CD4+ cells. By virtue of their induction of cytokines typical of CD4+ T-helper type 2 (Th2) cells-interleukin (IL)-4, 10, and 13-respiratory sensitizers may be identified and differentiated from contact sensitizers which induce Th1 cytokines (IL-2 and IFN-gamma). Our previous work suggested that the ribonuclease protection assay (RPA) was useful in identifying the respiratory sensitizer, trimellitic anhydride (TMA), based on quantitative differences in Th2 cytokine mRNA as compared to the contact sensitizer dinitrochlorobenzene (DNCB). Therefore, the purpose of the studies described in this report was to expand the chemicals tested in the RPA. To this end, four acid anhydrides with known respiratory sensitization potential, TMA, maleic anhydride (MA), phthalic anhydride (PA) and hexahydrophthalic anhydride (HHPA), were tested. Although previously determined to induce immunologically equivalent responses in a local lymph node assay (LLNA), the initial dose chosen (2.5%) failed to induce Th2 cytokine mRNA expression. To determine if the lack of cytokine expression was related to dose, LLNAs were conducted at higher doses for each of the anhydrides. The highest doses evaluated (four- to six-fold higher than those used in the initial RPA) gave equivalent proliferative responses for the various anhydrides and were used for subsequent RPA testing. At these higher doses, significant increases in Th2 versus Th1 cytokine mRNA were observed for all anhydrides tested. These results suggest that the RPA has the potential to serve as a screen for the detection of LMW airway sensitizing chemicals. However, the basis for selecting immunologically equivalent doses may require some modification.

Cytokine profiling for chemical sensitizers: application of the ribonuclease protection assay and effect of dose.

Exposure to chemicals in domestic and occupational settings may contribute to increases in asthma and allergy. Airway hypersensitivity (AHS) is T helper-2 (Th2) cell associated, whereas contact hypersensitivity (CHS) is T helper-1 (Th1) cell associated. The distinct cytokine profiles produced by these cells may provide a means of distinguishing respiratory sensitizers from contact sensitizers. In this study, female BALB/c mice were exposed twice on the flanks and three times on the ears using the airway sensitizer trimellitic anhydride (TMA) or the contact sensitizer dinitrochlorobenzene (DNCB). At various times following exposure, total mRNA was extracted from draining lymph node cells and cytokine mRNA profiles analyzed using a multiprobe ribonuclease protection assay (RPA). The Th2 cytokines IL4, IL10, and IL13 were significantly increased in response to TMA compared to DNCB, with optimal detection occurring 14 days following initial exposure. To determine its effect, dose was varied in flank exposures, ear exposures, or both simultaneously. When dose was varied during flank exposures only, TMA induced higher levels of Th2 cytokines than DNCB at all doses tested. DNCB did not induce Th1 cytokines at any dose tested. Variation of TMA dose during both exposures similarly induced Th2 cytokines. Dose only appeared to be a factor when TMA concentration was varied during the ear exposures alone. Thus, these studies suggest that quantitative differences in Th2 responses between TMA and DNCB may be demonstrated over a wide range of doses and these differences may be detected by RPA following dermal exposure to these sensitizers.

Subchronic inhalation toxicity of the chlorinated propane 1,1,1,3,3,3-hexachloropropane (HCC-230fa).

Male and female rats were exposed by inhalation (whole body) to HCC-230fa (1,1,1,3,3,3-hexachloropropane) for 6 h/day, 5 days/week over a 15-week period. Concentrations of 0, 0.50, 2.5, and 25 ppm were studied. A total of eight groups/sex were exposed. Four groups of male and four groups of female rats were used to measure clinical signs and growth, clinical pathology, and tissue pathology. The remaining four groups of male rats were used for immunotoxicological and sperm assessment evaluations, and the remaining four groups of female rats were used for immunotoxicological evaluation. Following the exposure period, surviving male rats were kept for a 1- or 3-month recovery period. Male and female rats exposed to 25 ppm had lower mean body weights, mean body weight gains, and food consumption during the exposure period. Male and female rats exposed to 25 ppm and sacrificed immediately after the exposure period had minimally decreased total leukocyte and lymphocyte counts. These changes were considered to be marginally adverse. Pathologic examination revealed hepatocellular hypertrophy in 0-day recovery males and an increased incidence and/or severity in chronic progressive nephropathy in 0-day, 1-month recovery, and 3-month recovery males at 25 ppm. No other pathological changes, including the testis, epididymis, and other accessory sex organs, were noted in rats during the study. Evaluation of sperm parameters at the end of the exposure period showed statistically significant decreases in epididymal sperm number per cauda epididymis, percent motile sperm, and percent normal sperm morphology at 25 ppm. The biological significance of the slight changes observed in the sperm parameters in the absence of histopathological changes is unclear. After a 1-month recovery period, no biologically significant differences in sperm parameters were noted at 25 ppm compared with controls. Exposure to HCC-230fa did not significantly alter the primary humoral immune response to sheep red blood cell (SRBC). Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) was considered to be 2.5 ppm.

90-day feeding and one-generation reproduction study in Crl:CD BR rats with 17 beta-estradiol.

Over the past several years, there has been increasing concern that chemicals and pesticides found in the environment may mimic endogenous estrogens, potentially producing adverse effects in wildlife and human populations. Because estrogenicity is one of the primary concerns, a 90-day/one-generation reproduction study with 17 beta-estradiol was designed to set dose levels for future multigenerational reproduction and combined chronic toxicity/oncogenicity studies. The purpose of these studies is to evaluate the significance of a range of responses as well as to provide benchmark data for a risk assessment for chemicals with estrogen-like activities. This 90-day/one-generation reproduction study was conducted in male and female Crl:CD BR rats using dietary concentrations of 0, 0.05, 2.5, 10, and 50 ppm 17 beta-estradiol. Endpoints were chosen in order to evaluate both subchronic and reproductive toxicity. In addition, several mechanistic/biochemical endpoints were evaluated for their usefulness in follow-up studies. In the P1 generation, dietary administration of 2.5, 10, and 50 ppm 17 beta-estradiol produced dose-dependent decreases in body weight, body weight gain, food consumption, and food efficiency. At 10 and 50 ppm 17 beta-estradiol, minimal to mild nonregenerative anemia, lymphopenia, decreased serum cholesterol (50 ppm only), and altered splenic lymphocyte subtypes were also observed in the P1 generation. Additionally, at these concentrations, there were changes in the weights of several organs. Evidence of ovarian malfunction, characterized by reduced numbers of corpora lutea and large antral follicles, was observed at 2.5 ppm 17 beta-estradiol and above. Other pathologic changes in males and females fed 10 and 50 ppm 17 beta-estradiol included centrilobular hepatocellular hypertrophy; diffuse hyperplasia of the pituitary gland; feminization of the male mammary glands; mammary gland hyperplasia in females; increased number of cystic follicles in the ovary; hypertrophy of the endometrium and endometrial glands in the uterus; degeneration of seminiferous epithelium; and atrophy of the testes and the accessory sex glands. In the reproduction portion of this study, rats fed 10 or 50 ppm 17 beta-estradiol did not produce litters. While there was no evidence that the 50 ppm treated rats mated, 33.3% of the rats fed 10 ppm mated but did not produce litters. No effects on mating and fertility indices were observed in rats fed 0.05 and 2.5 ppm 17 beta-estradiol. Pup weights at birth were statistically decreased relative to control in the groups fed 0.05 and 2.5 ppm 17 beta-estradiol. Weights of the rats in the 0.05 ppm group recovered by postnatal day 4 and remained similar to control throughout the remainder of the study. The mean gestation length of the 0.05 ppm group was slightly, albeit not statistically significantly, shorter (0.5 days) than that of the control group, which may have contributed to the decrease in birth weight of the 0.05 ppm group. In contrast, the weights of the F1 generation rats fed 2.5 ppm 17 beta-estradiol remained decreased relative to the control group throughout the study. Parental administration of 17 beta-estradiol did not alter anogenital distance in male or female pups. The onset of sexual maturation, as measured by day of preputial separation in males and day of vaginal opening in females, was delayed in male rats fed 2.5 ppm (by 8.2 days) and was hastened in female rats fed 0.05 and 2.5 ppm (by 1.6 and 8.8 days, respectively). The age at vaginal opening ranged from 26 to 37, 26 to 35, and 21 to 25 days for rats fed 0, 0.05, and 2.5 ppm 17 beta-estradiol, respectively. Hence, the range of age at vaginal opening was similar between the control and 0.05 ppm group. The organ weight and pathologic alterations observed in the adult F1 generation rats were similar to those observed in the P1 generation rats. (ABSTRACT TRUNCATED)

Further evaluation of the incorporation of an immunotoxicological functional assay for assessing humoral immunity for hazard identification purposes in rats in a standard toxicology study.

A previous study (Ladics et al., 1995) conducted in our laboratory using the known immunosuppressant agent, cyclophosphamide, indicated that a functional assay for assessment of humoral immunity may be conducted in rats in a standard toxicology study. The objective of this study was to further examine the feasibility of conducting an immunotoxicological assay for assessing humoral immunity in rats in a standard toxicology study using a chemical, carbon tetrachloride (CCl4), whose principal target organ of toxicity is not the immune system. Specifically, the previous study and this study were done to determine whether the conduct of an assay for assessing humoral immune function would affect standard toxicological endpoints. Male CD rats were untreated or dosed orally for 30 or 90 days, excluding weekends, with vehicle or 12.5 or 25 mg/kg CCl4. Six days prior to sacrifice, selected rats were injected intravenously with sheep red blood cells (SRBC) for assessment of humoral immune function. One day prior to necropsy, blood for hematological and clinical chemical measurements was collected from each rat. On the day of necropsy standard protocol tissues were collected, weighed, processed to slides, and later examined microscopically. One-half of each spleen was used to assess spleen cell numbers and quantitate lymphocyte subsets (Thelper; Tcyt/sup; total T- and B-cells) by flow cytometry. Serum was analyzed for anti-SRBC IgM antibody by using an enzyme-linked immunosorbent assay. Administration of 12.5 and 25 mg/kg CCl4 for 30 days decreased SRBC-specific serum IgM levels 42 and 45%, respectively, while 25 mg/kg CCl4 for 90 days increased SRBC-specific IgM levels by 50%. CCl4 did not alter splenic lymphocyte subset numbers nor the weight nor morphology of lymphoid organs. Exposure to 25 mg/kg CCl4 did increase liver weight and serum sorbitol dehydrogenase levels, as well as produce centrilobular fatty change. SRBC administration did not alter any hematological or clinical chemistry parameters, nor lymphocyte subset numbers. With the expected exception of the spleen (slight increase in number and size of germinal centers), administration of SRBC did not significantly alter the weights or morphology of routine protocol tissues. Furthermore, administration of SRBC did not mask the rather mild hepatotoxic effects of CCL4 exposure observed in this study. Based on these and previous findings, it appears that a functional assay for assessing humoral immunity may be conducted in animals on standard toxicology study without altering standard toxicological endpoints.

Assessment of the skin sensitization potential of topical medicaments using the local lymph node assay: an interlaboratory evaluation.

The murine local lymph node assay (LLNA) is a method for the predictive identification of chemicals that have a potential to cause skin sensitization. Activity is measured as a function of lymph node cell (LNC) proliferative responses stimulated by topical application of test chemicals. Those chemicals that induce a threefold or greater increase in LNC proliferation compared with concurrent vehicle controls are classified as skin sensitizers. In the present investigations we have evaluated further the reliability and accuracy of the LLNA. In the context of an international interlaboratory trial the sensitization potentials of six materials with a history of use in topical medicaments have been evaluated: benzoyl peroxide, hydroquinone, penicillin G, streptomycin sulfate, ethylenediamine dihydrochloride, and methyl salicylate. Each chemical was analyzed in the LLNA by all five laboratories. Either the standard LLNA protocol or minor modifications of it were used. Benzoyl peroxide and hydroquinone, both human contact allergens, elicited strong LLNA responses in each laboratory. Penicillin G, another material shown previously to cause allergic contact dermatitis in humans, was also positive in all laboratories. Streptomycin sulfate induced equivocal responses, in that this material provoked a positive LLNA response in only one of the five laboratories, and then only at the highest concentration tested. Ethylenediamine dihydrochloride dissolved in a 3:1 mixture of acetone with water, or in 4:1 acetone:olive oil (one laboratory), was uniformly negative. However, limited further testing with the free base of ethylene diamine yielded a positive LLNA response when applied in acetone:olive oil (AOO). Finally, methyl salicylate, a nonsensitizing skin irritant, was negative at all test concentrations in each laboratory. Collectively these data serve to confirm that the local lymph node assay is sufficiently robust to yield equivalent results when performed independently in separate laboratories and indicate also that the LLNA is of value in assessing the skin sensitization potential of topical medicaments.

A critique of the World Resources Institute's report "Pesticides and the immune system: the public health risks".

A recent World Resources Institute (WRI) report concluded that pesticides are a likely cause of immune suppression for millions of people throughout the world. The gravity of this conclusion motivated us to review the scientific evidence cited in the report. The predominant human evidence came from cross-sectional studies conducted in the former Soviet Union. These studies were difficult to evaluate due to incomplete reporting and had obvious limitations in terms of subject selection, exposure assessment,lack of quality control, statistical analysis, adequacy of the comparison group, and confounding. The toxicologic evidence was comprised mainly of acute high-dose studies in which the exposure conditions resulted in systemic toxicity. The relevance of these studies to effects at typical human exposure levels is questionable. We did not find consistent, credible evidence to support the conclusion of widespread pesticide-related immune suppression. Nonetheless, the WRI report is an important document because it focuses attention on a potentially important issue for future research and brings a substantial literature of foreign language studies to the attention of Western scientists.

Evaluation of the primary humoral immune response following exposure of male rats to 17beta-estradiol or flutamide for 15 days.

There is a concern that certain industrial chemicals found in the environment may mimic or antagonize endogenous hormones and adversely affect the endocrine as well as the immune system. The objective of this study was to determine if exposure of Crl:CD (SD)BR male rats to 17beta-estradiol (17beta-E2), an estrogen receptor agonist, or flutamide (FLUT), an androgen receptor antagonist, would significantly alter the primary IgM humoral immune response to sheep red blood cells (SRBC). This study was conducted in the context of a male in vivo Tier I battery designed to identify endocrine-active compounds (EACs). The Tier I male battery consists of organ weights coupled with a comprehensive hormonal assessment. Rats were dosed by the intraperitoneal route for 15 days with vehicle or 0.001, 0.0025, 0.0075, or 0.050 mg/kg/day 17beta-E2 or 0.25, 1, 5, or 20 mg/kg/day FLUT. Six days prior to termination, selected rats were injected intravenously with SRBC for assessment of humoral immune function. Spleen cell number and spleen and thymus weights were obtained. Serum was analyzed for anti-SRBC IgM antibody by using an enzyme-linked immunosorbent assay. At 0.050 mg/kg/day 17beta-E2, mean final body and absolute thymus weights were significantly decreased to 84 and 65% of control, respectively. 17beta-E2 did not significantly alter spleen weight, spleen cell number, or the primary IgM humoral immune response to SRBC. The no-observed-adverse-effect level (NOAEL) for immune system alteration was 0.050 mg/kg/day 17beta-E2 since the decrease in absolute thymus weight was judged to be secondary to the decrements in body weight. In the Tier I male battery, responses to 17beta-E2 included decreased absolute testis and epididymis weights, decreased relative accessory sex gland unit weights, hormonal alterations (decreased serum testosterone (T), dihydrotestosterone (DHT), and luteinizing hormone (LH), and increased serum prolactin and E2 levels). The lowest-observed-adverse-effect level (LOAEL) for the reproductive indices was 0.001 mg/kg/day 17beta-E2 based on the hormonal alterations seen at this level; no NOAEL was established. Exposure to FLUT did not significantly alter mean final body, spleen, or absolute thymus weights, spleen cell number, or the primary IgM humoral immune response to SRBC. A significant increase (118% of control) in relative thymus weight was observed at 20 mg/kg/day FLUT. The NOAEL for immune system alteration was 5 mg/kg/day FLUT based on the increased relative thymus weights that were judged to be compound-related. In the Tier I male battery, responses to FLUT included decreased absolute epididymis and relative accessory sex gland unit weights and hormonal alterations (increased serum T, DHT, E2, and LH, and decreased follicle stimulating hormone levels). The LOAEL for the reproductive indices was 0.25 mg/kg/day FLUT based on the hormonal alterations seen at this level; no NOAEL was established. Based on these data, the reproductive and not the immune system appears to be the primary target organ of toxicity in young adult male rats treated with either 17beta-E2 or FLUT.

Assessment of immunotoxicity by multiparameter flow cytometry.

Flow cytometry is a unique technology useful in the examination of effects of immunotoxic agents on target cells of the immune system. The purpose of this workshop was to provide an overview of the use of flow cytometry in new and established models of immunotoxicity, with emphasis on the potential applications, assay validation, and potential pitfalls. This overview begins with a discussion of methods useful in the assessment of Ca2+-dependent mechanisms of lymphoid cell activation in surface marker-defined human B cells, T cells, and monocytes. A discussion of the use of flow cytometry in analysis of apoptosis is also presented in this paper. The second paper presents data on the development and use of flow cytometry as an alternative to a Cr51 release assay for an assessment of cytotoxic T cell activation. The use of surface markers for characterizing and distinguishing the effects of chemical irritants from sensitizers is next presented, followed by an overview of the use of fluorescent probes to assess cell thiol status and overall oxidant-induced injury to lymphoid cells. Finally, an interlaboratory study designed to compare and evaluate the use of flow cytometry procedures in rat splenic cell subtyping is presented. Overall, these studies demonstrate the utility of flow cytometry assays in immunotoxicologic research, but further efforts are needed in the validation of many of these assays for routine use in immunotoxicologic testing.

Further evaluation of the local lymph node assay in the final phase of an international collaborative trial.

The local lymph node assay (LLNA) is a method used for the prospective identification in mice of chemicals that have the potential to cause skin sensitization. We report here the results of the second and final phase of an international trial in which the performance of the assay has been evaluated using seven test materials in five independent laboratories. The additional chemicals examined here included compounds which are considered less potent allergens than some of those tested in the first phase of the investigation, and includes hexylcinnamic aldehyde (HCA), a chemical recommended by the Organization for Economic Cooperation and Development (OECD) as a positive control for skin sensitization studies. In each laboratory all skin sensitizing chemicals examined (2,4-dinitrochlorobenzene {DNCB}, HCA, oxazolone, isoeugenal and eugenol) elicited positive responses of comparable magnitude as judged by the derived lowest concentration of test chemical required to elicit a 3-fold or greater increase in the proliferative activity of draining lymph node cells compared with vehicle-treated controls. We observed that sodium lauryl sulphate, considered to be a non-sensitizing skin irritant, also induced a positive response in the assay. Para-aminobenzoic acid (pABA), a nonsensitizing chemical, was negative at all test concentrations in each laboratory. Some laboratories incorporated minor modifications into the standard assay procedure, including the evaluation of lymph nodes pooled from individual mice rather than treatment groups and the use of statistical analyses. The use of statistics did not markedly change the determination of the lowest concentration yielding a positive response. These data confirm that the local lymph node assay is robust and yields equivalent results when performed independently.

An international evaluation of the murine local lymph node assay and comparison of modified procedures.

The murine local lymph node assay is a predictive test for the identification of skin-sensitizing chemicals. The method has been the subject both of national inter-laboratory studies and of extensive comparisons with guinea pig tests. In the investigations reported here, the local lymph node assay has been evaluated further in the context of an international study comprising five independent laboratories. In addition, the influence of minor modifications to the standard assay procedure on the performance of the test has been examined. The modified procedures investigated were exposure of mice for 4 rather than 3 consecutive days, excision of lymph nodes 4 rather than 5 days after the initiation of exposure and the use of an alternative isotope. All five laboratories, irrespective of whether the standard or a modified protocol was used, were able to identify accurately, and with comparable sensitivity, potassium dichromate and 2,4-dinitrochlorobenzene as skin sensitizers. Using standard criteria, none of the laboratories recorded positive responses with methyl salicylate, a non-sensitizer. In the standard protocol, lymph nodes are pooled for each experimental group and the vigor of responses measured as a stimulation index relative to vehicle controls. A stimulation index of 3 or greater is considered to indicate skin-sensitizing potential. One further modification adopted by three of the laboratories was to analyze nodes from individual animals and, thereby, permit statistical evaluation. This allowed a direct comparison of statistical significance with the conventional stimulation index as criteria for a positive response. The data indicate that, while statistical evaluation may provide, in some instances, for small increases in sensitivity, this may be at the expense of some loss of selectivity. There are, however, insufficient data presently to draw firm conclusions regarding the relative value of statistical analysis. These studies demonstrate that the local lymph node assay is sufficiently robust to accommodate minor procedural and technical modifications without material changes in test performance.

Possible incorporation of an immunotoxicological functional assay for assessing humoral immunity for hazard identification purposes in rats on standard toxicology study.

The objective of this study was to examine the feasibility of conducting an immunotoxicological assay for assessing humoral immunity in rats on standard toxicology study. Male CD rats were untreated or dosed intraperitoneally daily for 30 or 90 days, excluding weekends, with vehicle or 2 mg/kg cyclophosphamide (CY). Six days prior to sacrifice, selected rats were injected intravenously with sheep red blood cells (SRBC). One day prior to necropsy, blood samples for hematological and clinical chemical measurements were collected from each rat. On the day of necropsy standard protocol tissues were collected, weighed, processed to slides, and examined microscopically. One-half of each spleen was used to prepare a single cell suspension in order to assess spleen cell numbers. Serum was analyzed for anti-SRBC IgM antibody using an enzyme-linked immunosorbent assay. A second set of studies was performed to examine further the effect of SRBC administration on lymphoid organ weights using 30- and 90-day study age-equivalent naive male CD rats. Exposure of animals to 2 mg/kg CY for 30 or 90 days resulted in a 28% and 61% decrease, respectively, in SRBC-specific serum IgM levels. CY treatment also caused mild alterations in some leukocytic parameters, with significant decreases of 35% and 33% in white blood cell and lymphocyte counts, respectively, observed in 30-day CY-treated animals receiving SRBC. Injection of SRBC alone did not alter hematological or clinical chemistry parameters. With the expected exception of the spleen (increased number and size of germinal centers), administration of SRBC did not significantly alter the weights or morphology of routine protocol tissues. Furthermore, administration of SRBC did not mask the immunosuppressive effects of CY treatment under the conditions of this study. Based on our preliminary findings, a functional assay for assessing humoral immunity may be conducted in animals on standard toxicology study.