An early effect of aflatoxin B1 administered in vivo on the growth of bone marrow CFU-GM and the production of some cytokines in rats

Our data demonstrate the granulopoietic toxicity of aflatoxin B₁ (AFB₁)in vivo and show an impact of this mycotoxin on the production of some humoral regulatory factors dealing with the granulopoietic developmental pathway (CSA, IL-1, IL-2). The dose of AFB₁ studied represented approximately 1/5 of LD₅₀ for young male rats. An early suppressive effect of AFB₁ towards CFU-GM was transient in treated animals. The peak in granulopoietic activity was preceded in time by an increased CSA and IL-1 formation. Elevated IL-2 synthesis and increased T cell activation paralleled the peak in granulopoietic activity.Abbreviations AFB₁ Aflatoxin B₁ - CFU-GM granulocyte-monocyte colony-forming unit - CSA colony-stimulating activity - CSF colony-stimulating factor - GM-CSF granulocyte-monocyte CSF - G-CSF granulocyte CSF - M-CSF monocyte CSF - IL-1–6 Interleukin 1–6 - TNF tumour necrosis factor - IFN Interferon     

Effect of aflatoxin B1 on the delayed type hypersensitivity and phagocytic activity of reticuloendothelial system in chickens

Efforts were made to see the effect of feeding aflatoxin B₁ at 0.3 ppm level on various aspects of the immune system in chickens. The birds were fed aflatoxin B₁ (AFB₁) mixed ration from 0 to 6 weeks of age and thereafter normal feed was given up to 12 weeks of age. The delayed type hypersensitivity reaction of these birds was assessed by contact sensitivity to Dinitrofluorobenzene. The dietary AFB₁ significantly suppressed the cell mediated immune response at all the three periods tested (30, 45 and 60 days of age). The toxin showed residual effect on immunity as the suppression of cell mediated immunity was maximum three days after the withdrawal of toxin from the feed. The effect of AFB₁ on the phagocytic status of reticulo endothelial system (RES) was assessed by colloidal carbon clearance test at various intervals. The residual effect of toxin was observed on RES too as phagocytic index of AFB₁ fed birds was significantly lowered up to 45 days of age.     

Immunotoxicity of phosphamidon following subchronic exposure in albino rats

Effect of subchronic doses of phosphamidon exposure on humoral and cell mediated immune (CMI) responses were studied in male albino rats using SRBC, ovalbumin and KLH as antigens. Humoral immune responses were assessed by estimating antibody titre against antigen and splenic plaque forming cells (PFC) assay. CMI responses were studied by using leucocyte migration inhibition (LMI), macrophage migration inhibition (MMI) and delayed type hypersensitivity (DTH) response. Results obtained in the present study revealed marked suppression of humoral and CMI responses in a dose dependent pattern. Hence, suppression of immune responses by phosphamidon even at subchronic doses is clearly an important aspect for its safety evaluation.     

Preliminary determination of mycotoxin binding to soil when leaching through soil with water

Mycotoxin-contaminated crops that are left in the field are potential contaminants of groundwater. Aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁) distribution in soil-water systems and the comparative response of aflatoxin-contaminated corn and pure aflatoxin when leached through soil were investigated using columns. Each experiment was repeated once. Eluates and soil extracts were analyzed for AFB₁ and its metabolites and FB₁ along with different amounts of pure FB₁ and water mixtures. AFB₁ was detected in water samples from columns containing 10% and 20% silty clay loam soil and aflatoxin-contaminated corn mixtures and in the upper (top) 2.5 cm of soil from the 10 cm soil column. Aflatoxin B₂ (AFB₂) was detected in eluates from the column containing 10% soil and aflatoxin-contaminated corn mixture and from the column containing aflatoxin-contaminated corn alone. No AFB₂ was detected in eluates from the column containing 20% soil and aflatoxin-contaminated corn mixture. No detectable amount of aflatoxin was observed in eluates from the containing 50% silty clay loam soil and aflatoxin contaminated corn. No detectable amount of FB₁ was observed in eluates or soil extracts, but FB₁ was detected in the mixtures of pure FB₁ and water.     

Cytotoxicity of aflatoxin B1 and its chemically synthesised epoxide derivative on the A549 human epithelioid lung cell line

Aflatoxin B₁ (AFB₁) is a carcinogenic mycotoxin found in feeds and in airborne grain dusts. Aflatoxin B₁ requires biotransformation to the AFB₁-8,9 epoxide (AFBO) by a bioactivation system and subsequent covalent binding to DNA or proteins, to exert its carcinogenic potential. The lung contains cytochrome P₄₅₀, prostaglandin-H-synthase, lipoxygenase, epoxide hydrolase and other bioactivation enzymes, and is thus a potential target for the effects of AFB₁ via the routes of inhalation and ingestion. The A549 human epithelioid lung cell line and the methylthiazol tetrazolium (MTT) bioassay were used to investigate the cytotoxicity of AFB₁ and its chemically synthesised epoxide (AFBO) in vitro. Statistical analysis of the MTT results indicated that there were overall significant differences between the control and both the AFB₁-treated (p < 0.0001) and AFBO-treated cells (p = 0.00 2). However, there was no significant difference between AFB₁ and AFBO-treated cells, when the entire range of concentrations were assessed against each other (p = 0.2877). Whenanalysed at each concentration, only at 0.01 mM was there a significant difference between the effects of AFB₁ and AFBO (p = 0.0358). The results of this investigation show that AFB₁ and AFBO are both cytotoxic in the A549 cell line.This revised version was published online in October 2005 with corrections to the Cover Date.     

Effects of aflatoxin B1 on in vitro cultures ofNicotiana tabacum var. Samsun

Calli ofNicotiana tabacum (tobacco) were treated with two dose ranges of aflatoxin B₁ (0.1–2.0 µg ml^–1 - low does; 5–25 µg ml^–1 aflatoxin B₁). The ability of calli to recover following 3 weeks of toxin exposure was also investigated. The I₅₀ (50% inhibition) value for fresh mass accumulation was approximately 2 µg ml^–1 AFB₁. Fresh mass accumulation was significantly lower than the control value from 0.5 µg ml^–1 AFB₁. Following 3 weeks growth without a toxin source, the growth of calli up to and including 10 µg ml^–1 AFB₁, was significantly greater than control calli, indicating reversibility of the toxic effects. With increasing toxin concentration, chlorophyll content of callus was inhibited from 0.5 µg ml^–1. Transfer to a toxin-free medium resulted in a degree of recovery (up to 0.5 µg ml^–1). In the dose range 5–25 µg ml^–1, the levels of chlorophyll were drastically reduced, with no recovery following AFB₁ removal. Electron microscopy revealed a disruption of chloroplast structure as an early deteriorative event in AFB₁ exposure of callus cells. Protein levels were less sensitive, with inhibition manifested only in the high dose range. Shoot development occurred at all concentrations, but was significantly inhibited from 5 µg ml^–1 AFB₁. Recovery following toxin removal was minimal at these higher AFB₁ concentrations. The number of necrotic calli increased progressively from 5 µg ml^–1 as toxin levels increased.     

Naturally occurring chlorophyll derivatives inhibit aflatoxin B1-DNA adduct formation in hepatoma cells

The inhibitory effects of four chlorophyll derivatives (chlorophyllide [Chlide] a and b and pheophorbide [Pho] a and b) on aflatoxin B₁ (AFB₁)-DNA adduct formation, and on the modulation of hepatic glutathione S-transferase (GST) were evaluated in murine hepatoma (Hepa-1) cells. Enzyme-linked immunosorbent assay showed that pretreatment with Chlide or Pho significantly reduced the formation of AFB₁-DNA adducts, and that Pho was the most potent inhibitor. However, wash-out prior to adding AFB₁ totally eliminated inhibition by Childe and partially eliminated inhibition by Pho, indicating that the inhibitory effect of Chlide, and to some extent Pho, was mediated through direct trapping of AFB₁. Furthermore, spectrophotometric analysis showed that Pho treatment could increase GST activity in Hepa-1 cells. These observations indicate that the chlorophyll derivatives studied may attenuate AFB₁-induced DNA damage in the Hepa-1 cell by direct trapping of AFB₁. Pho provided additional protection not only by direct trapping, but also by increasing GST activity against hepatic AFB₁ metabolites.     

Neutral red (NR) assay for cell viability and xenobiotic-induced cytotoxicity in primary cultures of human and rat hepatocytes

Neutral red (NR) in medium was absorbed and concentrated in lysosomes of cultured rat and human hepatocytes. NR uptake increased with the time of incubation and reached a plateau in 2 hr. Uptake was proportional to the concentration of the NR solution and the numbers of viable liver cells. Prolonged culture of hepatocytes increased the numbers of lysosomes, and thus, the dye accumulation. The NR can be extracted from lysosomes for quantitative measurement of hepatocyte viability and cytotoxicity of xenobiotics. With this assay, several serum-free media (e.g., Waymouth's, MEM, LHC-8, etc.) were compared for the maintenance of viable hepatocytes in vitro. Interestingly, LHC-8 medium, which is used to grow human bronchial epithelial cells, best preserved viable rat hepatocytes. The cytotoxic effects of dimethylnitrosamine (DMN) and aflatoxin B₁ (AFB₁) were examined by NR assay on rat and human hepatocyte cultures and were found to be dependent on dose and time of the exposures. NR₅₀ was 20 mM for DMN and 0.072 µM for AFB₁ in rat hepatocytes with 24 hr of exposures and reduced to 12.5 mM for DMN and 0.053 µ uM for AFB₁ with 48 fr exposures. Human hepatocytes were more resistant to the toxicity of both chemicals; NR₅₀ values were 100 mM DMN and 1.8 µM AFB₁ respectively, for 24 hr treatments. Compared with lactate dehydrogenase (LDH) leakage test, the NR assay was simpler and more sensitive in determining the viability and cytotoxicity of xenobiotics in primary cultures of hepatocytes.Abbreviations NR Neutral Red - MEM Eagle's Minimum Essential Medium - DMN dimethylnitrosamine - AFB₁ aflatoxin B₁ - LDH lactate dehydrogenase - HBSS Hanks balanced salt solution; - EDTA ethylene bis (oxyethylenenitrilo)-tetraacetic acid - L-15 Leibovitz's 15 - NADH B-nicotinamide adenine dinu - FBS fetal bovine serum - IA immediate autopsy Contribution No. 2816 from Laboratory of Genotoxicology.     

Mycobiota in poultry feeds and natural occurrence of aflatoxins, fumonisins and zearalenone in the Rio de Janeiro State, Brazil

The intake of mycotoxin-contaminated feeds can lead to nutrient losses and may have adverse effects on animal health and on productivity. The aims of this study were (1) to determine the mycobiota present in poultry feed samples, and (2) to evaluate the natural occurrence of aflatoxin B₁, fumonisin B₁ and zearalenone. Fungal counts were similar between all culture media tested (10³ CFU g⁻¹). The most frequent genus isolated was Penicillium spp. (41.26%) followed by Aspergillus spp. (33.33%) and Fusarium spp. (20.63%). High precision liquid chromatography was applied to quantify aflatoxin B₁ and fumonisin B₁. Thin layer chromatography was used to determine zearalenone levels. Aflatoxin B₁ values ranged between 1.2 and 17.5 μg kg⁻¹. Fumonisin B₁ levels ranged between 1.5 and 5.5 μg g⁻¹. Zearalenone levels ranged between 0.1 and 7 μg g⁻¹. The present study shows the simultaneous occurrence of two carcinogenic mycotoxins, aflatoxin B₁ and fumonisin B₁, together with another Fusarium mycotoxin (zearalenone) in␣feed intended for poultry consumption. Many samples contained AFB₁ levels near the permissible maximum and it could affect young animals. A synergistic toxic response is possible in animals under simultaneous exposure.     

Interaction between aflatoxin B1 and oxytetracycline in isolated rat hepatocytes

Isolated rat hepatocytes were used as an in vitro model to investigate A possible interaction between oxytetracycline (OXT) and aflatoxin B₁ (AFB₁). LDH leakage, RNA and protein synthesis and glycogen accumulation were measured in the presence of both drugs, either separately or in combination. The evolution of LDH leakage during the incubation was identical in untreated and treated cells. AFB₁ inhibited RNA and protein synthesis at a concentration of 10^–7 M and 10^–6 M, respectively, and higher, whereas OXT did not influence RNA synthesis but inhibited protein synthesis at the highest tested concentration, 10^–3 M. As far as glycogen is concerned, rats were injected with glucagon before sacrifice in order to obtain a constant synthesis rate in isolated hepatocytes. AFB₁ inhibited the accumulation of glycogen from 10^–6 M upward. This effect was never observed before 90 min of incubation. OXT had no effect on glycogen synthesis. In the presence of both drugs, no interaction was demonstrated as far as RNA and protein synthesis were concerned, but OXT opposed the inhibition induced by AFB₁ on glycogen accumulation. If the in vivo protection, provided by OXT against AFBI-induced toxicity, is due to a direct interference in the toxic mechanisms of the mycotoxin, these results show that OXT does not influence the AFB₁-inhibition of RNA and protein synthesis. The latter are early and sensitive parameters inhibited by AFB₁. On the contrary, taking into consideration the results on glycogen accumulation, it seems more interesting to investigate further this metabolism.Abbreviations AFB₁ Aflatoxin B₁ - OXT Oxytetracycline - DMEM Dulbecco's Modified Eagle's Medium - LDH Lactate Dehydrogenase - DMSO Dimethyl Sulfoxide - BSA Bovine Serum Albumin     

Effect of dietary β-1,3 glucan on immune responses and disease resistance of healthy and aflatoxin B1-induced immunocompromised rohu (Labeo rohita Hamilton)

The immunostimulant β-1,3 glucan was fed at 0·1% in feed for 7 days to healthy and aflatoxin B₁(AFB₁)-induced immunocompromised fish, Labeo rohita (one of the major tropical carp species), in a 60 day trial. The effects of AFB₁, glucan and their interactions on non-specific and specific immunity levels and disease resistance of fish were studied. A single intraperitoneal injection of AFB₁at 1·25 mg kg⁻¹body weight) caused a significant (P< 0·05) reduction in non-specific immunity as measured through neutrophil phagocytic indices, serum bactericidal activity, and specific immunity as measured through bacterial agglutination titre against Edwardsiella tarda, as well as reduced protection against Aeromonas hydrophila challenge in comparison to control fish which were exposed neither to aflatoxin nor to glucan. Feeding of glucan to healthy fish raised the non-specific and specific immunity level and protection against bacterial infection compared with the control. Feeding of glucan to AFB₁-induced immunocompromised fish for 7 days significantly raised the degree of resistance against A. hydrophila challenge and the non-specific immunity level in comparison to non-treated AFB₁exposed fish. Although feeding of glucan was able to increase specific immunity, al measured through haemagglutination titre against sheep red blood cells, and bacterial (E. tarda) agglutination titre in healthy fish in comparison to all other groups, no significant increase in specific immunity to the aflatoxin-exposed group was seen.     

A novel glass fiber disc culture system for testing of small amounts of compounds on growth and aflatoxin production byAspergillus flavus

A new method for growingAspergllius flavus for experimental studies is presented. The system consists of a humidified vial with a thick septum pierced by a pin on which a glass fiber disc is affixed. The disc contains the test solution and inoculum plus medium. The method has been used to assess the effect of variations in culture conditions on production of aflatoxin B₁ (AFB₁). The AFB₁ level was affected by the amount of medium placed on the disc and type of disc material. The results for different types of glass fiber and quartz discs were compared with AFB₁ produced by fungus grown in liquid medium or on paper discs. When compared to a liquid medium culture there was a 15 to 20-fold increase in AFB₁ for one type of disc. Incubations with less than 14 µl of medium gave satisfactory results. A crude phosphatidylcholine preparation at a concentration of 0.7% of the medium resulted in a 4-fold increase in AFB₁.Abbreviations AFB₁ aflatoxin B₁ - CV coefficient of variation - PC phosphatidylcholine - SD suspended disc     

The effects of aflatoxin B1 on growth hormone regulated gene-1 and interaction between DNA and aflatoxin B1 in broiler chickens during hatching

Many types of aflatoxin cause problems for both public and animal health. Aflatoxin B₁ (AFB₁) is the most toxic and commonly encountered fungal toxin that appears in poultry feed and in feeds stored under unsuitable conditions. AFB₁ decreases feed quality, egg production and fertility of hatching eggs. Also, AFB₁ alters the development of embryos by infecting eggs. We investigated using sequence analysis the changes caused by different concentrations of AFB₁ on the promoter sequences of the growth hormone regulated gene-1 (GHRG-1) in chick embryo at 13, 17, 19 and 21 days incubation. DNA isolated from the liver of chick embryos treated with different concentrations of AFB₁ was separated using agarose gel electrophoresis to detect apoptosis, and DNA interaction with AFB₁ was investigated using plasmids to detect changes in electrophoretic mobility and their effects on DNA. Base changes of the promoter sequences of GHRG-1 in 5 ng/egg, 15 ng/egg and 40 ng/egg doses of AFB₁ were increased on day 19 compared to base changes of the same AFB₁ doses on day 13. We also found that AFB at different concentrations changed the mobility of DNA by binding to it, and that high doses of AFB1 destroyed DNA. The DNA interaction study using plasmid demonstrated that AFB₁ at high doses was bound to plasmid DNA, slowed its mobility and inhibited restriction cuts.     

Aflatoxin B1-induced ultrastructural alterations in matureZea mays embryos

Mature maize (Zea mays L.) embryos were exposed to aflatoxin B₁ (AFB₁) concentrations ranging from 0.1 to 25 µg/ml for 9 days. With increasing toxin concentration above 2 µg/ml, primary root elongation of germinated embryos was progressively inhibited, to reach a maximum value of 81% at 25 µ/ml toxin. An ultrastructural investigation of the subcellular alterations induced following toxin exposure provided evidence of deteriorative changes in several compartments of the plant cell. Alteration in membrane integrity (e.g., the tonoplast, plasmalemma and inner mitochondrial membrane) was a frequent feature of many cells. Apparent fusion of vacuoles, incorporation of cytoplasmic components into vacuoles and intravacuolar membrane whorls might be interpreted as deteriorative alterations. The results are discussed in the light of ultrastructural findings for other plant systems exposed to similar AFB₁ concentrations, as well as findings for animal systems.     

Lactobacillus rhamnosus Strain GG Modulates Intestinal Absorption, Fecal Excretion, and Toxicity of Aflatoxin B1 in Rats

In this study, the modulation of aflatoxin B₁ (AFB₁) uptake in rats by administration of the probiotic Lactobacillus rhamnosus GG was demonstrated. Fecal AFB₁ excretion in GG-treated rats was increased via bacterial AFB₁ binding. Furthermore, AFB₁-associated growth faltering and liver injury were alleviated with GG treatment.     

In vivo covalent binding of aflatoxin B1 and aflatoxin M1 to liver DNA of rat,mouse and pig

[¹⁴C]Aflatoxin B₁ (AFB₁) was isolated from cultures of Aspergillus parasiticus grown on [1-¹⁴C]sodium acetate. Covalent binding of AFB₁ to liver DNA of rat and mouse was determined 6–8 h after oral administration. The effectiveness of covalent binding, expressed as DNA binding per dose in the units of a ‘Covalent Binding Index’ (CBI), (μmol aflatoxin/mol DNA nucleotides)/(mmol aflatoxin/kg animal), was found to be 10 400 for rats and 240 for mice. These CBI partly explain the different susceptibility of the two species for the incidence of hepatic tumors.The corresponding values for pig liver DNA, 24 and 48 h after oral administration, were found to be as high as 19 100 and 13 300. DNA-binding has not so far been reported for this species although it could represent an appropriate animal model for studies where a human-like gastrointestinal tract physiology is desirable.Aflatoxin M₁ (AFM₁) is a metabolite found in the milk of cows that have been fed AFB₁-contaminated diet. [¹⁴C]AFM₁ was also found to be produced by cultures of A. parasiticus giving a yield of about 0.3% of the total aflatoxins. A test for covalent binding to rat liver DNA revealed a CBI of 2100 showing that AFM₁ must also be regarded as a strong hepatocarcinogen. It is concluded that AFB₁ contaminations should be avoided in dairy feed.     

A comparative evaluation of aflatoxin B1 genotoxicity in fish models using the Comet assay

Aflatoxin B₁ (AFB₁) is classified as a Group I hepatocarcinogen in humans by the International Agency for Research on Cancer (IARC). The alkaline Comet assay is a simple and rapid method by which DNA damage can be demonstrated as a function of tail moment. The present work is the first to evaluate the genotoxicity of AFB₁ in fish using the Comet assay. Two different species of fish were selected as models due to previously established sensitivity to AFB₁: rainbow trout (sensitive) and channel catfish (resistant). Fish were i.p. injected with 0.5 mg AFB₁/1 ml DMSO/1 kg body weight. The Comet assay was performed after 4 and 24 h on whole blood, liver, and kidney cells of both species. Trout blood and kidney tissue tested displayed significant (p<0.05) and extensive DNA damage (shown by increased tail moment) after 4 h which then decreased by 24 h. In liver cells, damage progressively increased over time. Conversely, similarly treated catfish showed no elevation in DNA damage over controls at the same doses. These results suggest that the Comet assay is a useful tool for monitoring the genotoxicity of mycotoxins such as AFB₁ and for evaluating organ specific effects of these agents in different species.     

Effect of aflatoxin B1 on CD3 T cells andalkaline phosphatase in the intestine of mice

The aim of the study was to determine the effect of repeated applications of aflatoxin B₁ (AFB₁) on immunocompetent cells (CD3 T cells) and alkaline phosphatase in the intestinal mucosa. Mice were orally treated with AFB₁ for 24 days. The mucosa of the intestine showed a significant decrease in the number of CD3 T cells and a significantly lower level activity of alkaline phosphatase on day 24 in AFB₁ treated mice. Similarly, with changes in the small intestine, qualitative haematological parameters were modified in systemic immunity as lymphopenia, and neutropenia, monocytopenia. AFB₁ treated animals showed reduction in body weight gain and increased liver weight. We supposed that changes found in the small intestine are secondary to primary systemic haematological lesions. The decrease in CD3 T cells suggests a connection with the decrease in the host's resistance to infectious diseases. This revised version was published online in June 2006 with corrections to the Cover Date.     

Aflatoxins,discolouration and insect damage in dried cowpea and pigeon pea in Malawi and the effectiveness of flotation/washing operation in eliminating the aflatoxins

Aflatoxin contamination and biodeterioration were examined in 302 samples of dry cowpeas and pigeon peas that were randomly purchased from 9 districts of the Southern Region of Malawi during July and November 2015. Further, the impact of flotation/washing on aflatoxin levels on the pulses was elucidated. Aflatoxin analyses involved immunoaffinity column (IAC) clean-up and HPLC quantification with fluorescence detection (FLD) while legume biodeterioration assessments were done by visual inspection. Aflatoxins were frequently detected in cowpea (24%, max., 66 μg/kg) and pigeon pea (22%, max., 80 μg/kg) samples that were collected in the month of July. Lower aflatoxin incidence of 15% in cowpeas (max., 470 μg/kg) and 14% in pigeon peas (max., 377 μg/kg) was recorded in the November collection. Overall, aflatoxin levels were significantly higher in the pulses that were collected in November. However, there were no significant differences in the total aflatoxin (aflatoxin B₁ (AFB₁) + AFB₂ + AFG₁ + AFG₂) levels between the two types of pulses. Remarkably, in 76.2% of the aflatoxin positive cowpea and in 41.7% of the aflatoxin positive pigeon pea samples, aflatoxin G₁ concentration exceeded aflatoxin B_1. Insect damage percentage averaged at 18.1 ± 18.2% (mean ± SD) in the cowpeas and 16.1 ± 19.4% in pigeon peas. Mean discolouration percentage (number of pulses) of the cowpeas and pigeon peas was found to be at 6.7 ± 4.9 and 8.7 ± 6.2%, respectively. Washing and discarding the buoyant fraction was highly efficient in reducing aflatoxin levels; only 5.2 ± 11.1% of the initial aflatoxin level was found in the cleaned samples. In conclusion, cowpeas and pigeon peas sold on the local market in Malawi may constitute a hazard especially if floatation/washing step is skipped.     

Mechanisms of genetic control of immune responses

We examined multiple genetically regulated Immoral and cell-mediated immune (CMI) responses to poly(glu⁶⁰ala³⁰tyr¹⁰) (GAT) using a panel of mouse strains. We show that assignment of responder/nonresponder status depends upon the assay method. In addition, two distinct categories of nonresponder mice were found: (1) those which are unresponsive by all parameters tested (H-2 ^q and H-2 ^s haplotypes) and (2) those which are partially nonresponsive [H-2 ^bm12 mutant strain—a low/nonresponder by splenic plaque-forming cell (PFC) and delayed-type hypersensitivity (DTH) responses, but exhibits B6 parental levels of high GAT-specific T-cell proliferation (Tprlf) and interleukin-2 production]. The distinction between these two nonresponder types was confirmed by complementation tests in which significant GAT-specific PFC and DTH responses were seen in (H-2 ^q × H-2 ^bm12)F₁ hybrids, but not in (H-2 ^q × H-2 ^s )F₁ hybrids. Suppressor T cells (Ts) also play a selective role in nonresponsiveness to GAT. Cyclophosphamide treatment of nonresponders (to eliminate Ts activity) as well as immunization with GAT coupled to the immunogenic carrier MBSA result in the development of GAT-specific humoral, but not CMI responses. Our results indicate that the T cell is the cellular site of Ir gene expression and that Tprlf responses do not correlate with functional helper T-cell activity and suggest distinct, multi-step Th/Ts regulatory pathways in the development of humoral and CMI effector functions.