Immunomodulatory effects of the herbicide propanil on cytokine production in humans: In vivo and in vitro exposure

Abstract

Propanil, 3,4-dichloropropionanilide, a commonly used herbicide, has been shown to induce effects on the mouse immune system. The aim of this study was to assess the immunotoxicity of propanil in occupationally exposed agricultural workers and to characterize its molecular mechanism of action.

Seven agricultural workers intermittently exposed to propanil and 7 healthy matched controls entered the study. Data were collected through physical examination, and laboratory investigations addressed at the main serum, cellular, and functional immune parameters. The levels of exposure were assessed by determining the urine concentration of the major propanil metabolite, 3,4-dichloroaniline. The investigation of serum, cellular, and functional immune parameters suggested that propanil exposure results in a modest immunomodulatory effect, characterized by an increase in the plasma level of IgG₁ and in LPS-induced IL-6 release and, by a reduction in PHA-induced IL-10 and IFN release, associated with a reduced IFN/IL-4 ratio.

As observed, following in vivo exposure, in vitro treatment of human peripheral blood leukocytes with propanil resulted in a dose-dependent reduction in PHA-induced IFN-gamma and IL-10 production, while LPS-induced TNF-α production was not affected indicating a direct effect of propanil on selected immune parameters. We demonstrated that propanil interfering with PHA-induced intracellular calcium increase modulated IL-10 and IFN-gamma transcription and translation, which indicates that propanil acts on early events triggered by PHA.

Overall, our results suggest that human exposure to propanil has slight immunomodulatory effects, and point out that the inhibition of the PHA-induced intracellular calcium rise is an important target of propanil. These findings improve our understanding of the mechanism underlying propanil-induced immunotoxicity.

Introduction

Pesticides are designed to interfere with living species, and are necessarily characterized by variable levels of toxicity. Data are widely available suggesting that the immune system may be a target of the toxic effect of pesticides (Barnett and Rodgers, 1994, Repetto and Baliga, 1996).

Propanil (3,4-dichloropropionanilide) is an amide herbicide, extensively used on rice as a selective post-emergence control of a broad spectrum of weeds. Amide herbicides are photosynthesis inhibitors and propanil is a strong inhibitor of the Hill reaction (Yih et al., 1968a), the light-initiated reaction that splits water, resulting in the production of free oxygen by plants. With an environmental half life ranging from 1 to 3 days (Wauchope et al., 1992, Weed Science Society of America, 1994) and being easily degraded to 3,4-dichloroaniline (3,4-DCA) (Yih et al., 1968b, Bartha and Pramer, 1967), propanil shows a low persistence in the soil after application.

Propanil can be absorbed into the body through the respiratory tract, the digestive apparatus and the intact skin (Extoxxnet, 1993). After absorption by mammals, propanil is rapidly hydrolyzed, mainly to 3,4-DCA, by the liver enzyme acylamidase (Williams and Jacobson, 1966). In experimental animals, the half life of the compound is quite rapid irrespective the route of entry. In fact, after a single dose, the complete elimination from the body is within 48 to 72 h (Izmerov, 1984). No half-life data for humans are available.

Previous research in rodents has demonstrated that propanil may affect the immune system (Barnett and Gandy, 1989). Acute in vivo exposure to propanil in mice has shown that both propanil and its metabolite, 3,4-DCA, induced splenomegaly and thymic atrophy when given intraperitoneally or orally (Barnett et al., 1992). Experimental studies have established that exposure to propanil can inhibit the function of a variety of immune cells, including T helper cells, Natural killer cells, and macrophages. In addition, T-dependent and T-independent antibody production, contact hypersensitivity reaction, mixed lymphocyte reaction, and cytokine production, can be mentioned among the functional immune responses affected by propanil (Barnett and Gandy, 1989, Bartha and Pramer, 1967, Xie et al., 1997, Zhao et al., 1998). Despite a quite normal response to primary stimulation of CTL effectors in animals exposed to propanil, recent research study has shown that the secondary response resulted in complete abrogation of CTL lytic function and a delayed but significant long-term effect on CTL responsiveness (Sheil et al., 2006). Relative to cytokine production, it has been shown that propanil reduces TNF-α, IL-6, granulocyte-macrophage colony stimulating factor protein and message production in peritoneal and splenic macrophages, and IL-2 and γ-IFN production in splenocytes and T cell lines (Xie et al., 1997, Zhao et al., 1998).

In humans, as reported by the International Occupational Safety and Health Information Centre (http://www.ilo.org) and by the EPA (http://www.epa.gov), short-term high exposure to propanil may cause adverse effects in central nervous system (headache, confusion, drowsiness, dizziness) and blood, resulting in the formation of methaemoglobin. Locally, the compound may also cause irritation of skin, eyes and respiratory tract. Data on the immunotoxicity of propanil in humans are lacking. A study aimed to compare immune function parameters in children and adults living near rice fields with a matched control population assessed before, during, and after the agricultural season when propanil had been applied to crops, failed to show significant differences (McClure et al., 2001). This suggests that individuals living in the proximity of propanil-treated rice fields are not at increased risk of altered immune function. Even if propanil was detected in areas adjacent to rice fields treated with the herbicide, and even in the proximity of the houses, no propanil was detected in the studied homes, and no biological data supporting the hypothesis that propanil absorption took place have been collected (Richards et al., 2001). Therefore, the hypothesis that the population under study had not been exposed to the herbicide cannot be firmly ruled out. No studies on the effects of propanil on the immune system of occupational exposed workers are available. Due to the lack of information, we designed a study to evaluate the immune system of a group of agricultural workers occupationally exposed to propanil and to characterize, in vitro, the molecular mechanism of action.