Abstract

Because of the wide use of pesticides for domestic and industrial purposes, the evaluation of their potential effects is of major concern for public health. The myelotoxicity of the herbicide propanil (3,4-dichloroproprioanilide) and its metabolite 3,4-dichloroaniline (DCA) is well documented in mice, but evidence that pesticides may severely compromise hematopoiesis in humans is lacking. In this study, an interspecies comparison of in vitro toxicity of these two compounds on murine and human burst- and colony-forming unit-erythrocyte (BFU-E, CFU-E) and colony-forming unit–granulocyte/macrophage (CFU-GM) progenitors, has been carried out. Murine bone marrow progenitors and human cord blood cells were exposed to propanil or DCA in doses ranging from 10 μM to 1000 μM, and the toxic effect was detected by a clonogenic assay with continuous exposure to the compounds. The results on murine cells indicate that the erythrocytic lineage is the most sensitive target for propanil and DCA. On the other hand, human progenitors seem to be less sensitive to the toxic effects of both compounds than murine progenitors at the same concentrations (IC50 values are 305.2 ± 22.6 μM [total erythroid colonies] and >500 μM [CFU-GM] for propanil). Propanil was significantly more toxic to human erythroid progenitors than to human CFU-GM progenitors, as was found for the murine cells, emphasizing the role of the heme pathway as the target for propanil. These data confirm the evidence that the compounds investigated interfere with erythroid colony formation at different stages of the differentiation pathway and have different effects according to the dose.

Bone marrow, with its rapidly renewing cell populations, is one of the most sensitive tissues to cytotoxic agents (Bloom, 1993). Erythropoiesis is the part of hematopoiesis responsible for red blood cell production by cell proliferation and differentiation of specific erythroblastic progenitors, the burst- and colony-forming unit–erythrocyte (BFU-E and CFU-E). Defective erythropoiesis can be accompanied by bone marrow aplasia or hyperplasia or can be without significant bone marrow abnormality (Amess, 1993). Studies in laboratory animals have shown that myelotoxicity can be elicited at exposure levels that cause no other overt signs of toxicity in these animals (Hong et al., 1991).

Because of the wide use of pesticides for domestic and industrial purposes, the evaluation of their effects is of major concern for public health (Vial et al., 1996). Human exposure to these contaminants, especially if chronic, represents a risk for the immune system and predisposes to the onset of different pathologies.

Propanil (3,4-dichloropropionanilide) is a contact postemergence herbicide used extensively to control broad-leaved and grassy weeds, mainly in rice (Kimbrough, 1980).

Several works showed the immunotoxic potential of this compound on mammalian systems, with a depression of natural killer cell function and an alteration in macrophage function (Barnett and Gandy, 1989), whereas recent studies indicate that individuals living next to rice fields are not at increased risk of altered immune function due to propanil exposure (McClure et al., 2001).

Propanil is not persistent and is easily metabolized by rice and mammals under natural conditions (Chow and Murphy, 1975). Two of these metabolites, 3,4-dichloroaniline (DCA) and 3,3′,4,4′-tetrachloroazobenzene, are more toxic and more persistent than the parent compound (De Silva and Bodinayake, 1997). Long-term exposure to propanil results in red blood cell toxicity (WHO, 1993), whereas methemoglobinemic activity in animals (Singleton and Murphy, 1973) and humans has been shown after an acute exposure (Kimbrough, 1980).

This latter effect seems to be dependent upon the metabolism of propanil in the liver to DCA via an acylamidase-catalyzed hydrolysis of the parent compound (Singleton and Murphy, 1973), and further to N-hydroxy-3,4-dichloroaniline, which then enters the erythrocytes and is oxidized by hemoglobin to 3,4-dichloronitrosobenzene and methemoglobin (McMillan et al., 1990, 1991).

Ex vivo data on murine propanil myelotoxicity are available (Blyler et al., 1994), but evidence that pesticides may severely compromise hematopoiesis in human is lacking.

This work has been designed to evaluate the toxic effect of propanil or its metabolite DCA on CFU-E, BFU-E, and colony-forming unit–granulocyte/macrophage (CFU-GM) progenitors from human cord blood. An interspecies comparison was done with bone marrow cells obtained from CD-1 mice.

The xenobiotic effect on the progenitors has been detected by a clonogenic method, with continuous exposure to the compounds during the assay performance (Gribaldo et al., 1996; Noble and Sina, 1993; Tepperman et al., 1974)

MATERIALS AND METHODS

Chemicals.

Propanil (3-4-dichloropropionanilide, MW 218.09) and DCA (3,4-dichloroaniline, MW 162.02) were provided by Dr. Ehrenstorfer GmbH (Augsburg, Germany). Stock solutions of the test compounds were prepared in ethanol and then stored at –20°C. The final concentration of ethanol never exceeded 0.1%.

Source of human progenitor cells.

As the source of progenitors for the CFU-assays, human mononucleated cord blood cells were used. The cells were obtained, frozen, from Poietic Technologies, Inc. (Gaithersburg, MD, USA) and thawed before using. Briefly, 1 ml of cells was rapidly thawed in a water bath at 37°C and diluted in 1 ml of 2.5% human albumin (Sigma-Aldrich S.r.l., Milano, Italy), 5% Dextran 40 (Pharmacia Biotech Italia, Cologno Monzese, Italy), Iscove’s Modified Dulbecco’s Medium (IMDM, Gibco Life Technologies Italia S.r.l., San Giuliano Milanese, Italy) 0.22 μm filtered solution, and 8 ml of 10% fetal bovine serum (FBS, Gibco Life Technologies, San Giuliano Milanese, Italy), 3 U DNase/ml (Boehringer Mannheim Italia S.p.A., Monza, Italy) IMDM 0.22 μm-filtered solution. After 10 min, the solution was centrifuged at 800 × g for 10 min at 18–20°C. The pellet was then diluted in 30% FBS-IMDM, counted in a hemocytometer, and used for the clonogenic test at a concentration of 106 cells/ml.

Isolation of murine bone marrow cells.

This procedure was performed in rigorously sterile conditions on untreated mice. Three-week old CD-1 male mice weighing 14–16 g were purchased from Charles River Italia (Como, Italy). Following animal sacrifice by cervical dislocation, intact femurs were isolated by cutting muscle ligaments, cleaned, and placed in 100-mm Petri dishes containing 10 ml ice-cold IMDM supplemented with antibiotics (penicillin 100 U/ml and streptomycin 100 mg/ml; Sigma-Aldrich S.r.l., Milano, Italy). The ends of each femur were cut just below the head and bone marrow was flushed with 3 ml of IMDM without antibiotics. A single-cell suspension was produced by gently and repeatedly drawing the marrow cells through a syringe fitted with a 23-gauge needle. Bone marrow cells were then filtered through a 100-mm cell strainer and washed by centrifugation at 400 × g for 10 min at 20°C.

The pellet was resuspended in medium (3 ml IMDM + 30% fetal calf serum [FCS]) and 10 ml of cells was diluted with 90 ml Trypan blue and counted in a hemocytometer. Viability was usually 95% or greater, with no differences between experimental groups. Dilutions of the original cell suspension were made to achieve the correct number of cells per milliliter for the assay to be performed.

Human BFU-E/CFU-E assay.

Mononucleated cells, thawed as previously described, were seeded in MethoCult-H4230 (StemCell Technologies, Vancouver, BC, Canada). The medium was minus colony-stimulating factor (CSF) and erythropoietin, but contained FBS (30%), bovine serum albumin (BSA, 1%), methylcellulose (0.9%), 2-mercaptoethanol (10−4 M), and glutamine (2 mM). Cytokine stimulation was provided by the addition of 2.5 units/ml human recombinant erythropoietin (Boehringer Mannheim) to the tube containing 4 ml methylcellulose before the addition of compounds and cells.

The clonogenic assay was performed by adding 100 μl of 44X compound solutions (in IMDM) and 300 μl of cells (1.5 × 106 cells/ml) directly to a 4-ml methylcellulose tube. The final concentrations of compounds were 10, 20, 50, 100, 200, and 500 μM. Finally, 1 ml methylcellulose-cell suspension was seeded in 35-mm dishes, and the cultures were incubated at 37°C in 5% CO2 for 7 and 15 days.

Human CFU-GM assay.

Cord blood cells were seeded in MethoCult-H4534 (StemCell Technologies, Vancouver, BC, Canada). This medium contains CSF, without erythropoietin, and contains methylcellulose (1%), FBS (30%), BSA (1%), 2-mercaptoethanol (10−4 M), glutamine (2 mM), interleukin (IL)-3 (10 ng/ml), GM-CSF (10 ng/ml), and stem cell factor (SCF) (50 ng/ml). One hundred microliters of 44X compound solution (in IMDM) and 300 μl of cells (1.5 × 106 cells/ml) were added in tubes containing 4 ml of methylcellulose, and 1 ml of methylcellulose-cells suspension was seeded in 35-mm Petri dishes. The final concentrations of compounds were 20, 50, 200, 300, 400, and 500 μM. Cultures were incubated at 37°C in 5% CO2 for 14 days.

Murine BFU-E/CFU-E and GM-CFU assay.

Murine progenitors, collected as previously described, were washed, diluted in 30% FBS-IMDM, and then seeded in MethoCult-M3334 H4534 (StemCell Technologies, Vancouver, BC, Canada) for the BFU-E/CFU-E assay or in MethoCult-M3534 H4534 (StemCell Technologies, Vancouver, BC, Canada) for the GM-CFU assay. These media are specific for murine cells and contain methylcellulose (1%), FBS (15%), BSA (1%), bovine pancreatic insulin (10 μg/ml), human transferrin iron-saturated (200 μg/ml), 2-mercaptoethanol (10−4 M), and glutamine (2 mM). Stimulation of the erythroid lineage was obtained by the addition of erythropoietin (3 units/ml), whereas MethoCult-m3534 contained IL-3 (10 ng/ml), IL-6 (10 ng/ml), and SCF (50 ng/ml) to stimulate GM-CFU growth. The procedure was similar to that followed for human assays, with some modifications. The final concentrations of compounds were as follows: propanil 10, 20, 50, 100, 200, 500, and 1000 μM; DCA 10, 20, 50, 100, 200, 500, and 1000 μM. Finally, 1 ml methylcellulose-cell suspension was seeded in 35-mm dishes, and the cultures were incubated at 37°C in 5% CO2 for 3 and 10 days (BFU-E/CFU-E cultures) or 7 days (GM-CFU cultures).

Scoring of colonies.

CFU-E and BFU-E were scored after 7 and 15 days of incubation, respectively, for human cells, and after 3–5 and 8–10 days, respectively, for murine cells, using an inverted microscope with magnification ×25. A CFU-E colony was defined as an isolated single colony containing from 5 to approximately 100 well-hemoglobinized cells, whereas a BFU-E colony was defined as 3–8 closely spaced erythroid colonies of well-hemoglobinized cells.

Morphologically, three classes of CFU-E/BFU-E colonies can be observed: red, colorless, and heterogeneous colonies. Red cell colonies contain only red-pigmented cells; colorless colonies contain no pigmented cells; and heterogeneous colonies present yellow-brown-pigmented cells, red-pigmented cells, and nonpigmented cells.

Human CFU-GM colonies were scored after 14 days of incubation, whereas murine CFU-GM were scored after 7 days of incubation, using an inverted microscope. A CFU-GM colony was defined as an aggregate containing 50 or more cells (Pessina et al., 2001).

Morphologically, four classes of CFU-GM colonies can be observed: compact, diffuse and spread, multicentric, and multifocal colonies. A compact colony presents a central dense nucleus and a peripheral halo. Diffuse and spread colonies are without an apparent nucleus. A multicentric colony appears with two or more dense nuclei nearby, with a common peripheral halo growing at the same depth in the plate. Multifocal colonies are aggregates of several colonies or clusters with or without a peripheral halo (Rio et al., 1997a, b).

Data analysis.

Cell proliferation is expressed as a percentage of growth, with 100% corresponding to the number of colonies in the control dishes. The concentrations that inhibited 50% of growth (IC50) were interpolated according to the formula of Reed and Muench (1938). Data were expressed as mean ± SE of at least three experiments carried out in triplicate. Statistical analysis was performed by ANOVA followed by post-ANOVA tests (Fisher PLSD and Scheffe F-test). Values of p < 0.05 were considered statistically significant.

RESULTS

The toxic effect of propanil or its metabolite DCA on CFU-E, BFU-E, and CFU-GM colony formation from human cord blood progenitor cells and from bone marrow cells obtained from untreated mice was evaluated after direct exposure to the toxicants.

Propanil and DCA concentration-response curves obtained in three different assays on murine progenitor cells are presented in Figures 1, 2, and 3, respectively, and the relative IC50 values are reported in Table 1. Both propanil and DCA reduced the number of BFU-E, CFU-E, and CFU-GM colonies in a concentration-dependent fashion. As clearly shown by IC50 values, the erythroid progenitors were about 2.5 to 13 times more sensitive than the granulocyte-macrophage progenitors to the parent compound and its metabolite. With regard to the in vitro myelotoxic potential of the test compounds toward the erythroid lineage, in murine cells DCA was a more potent inhibitor of CFU-E and BFU-E colony formation than propanil.

Figures 4 and 5 represent the toxicity of the pesticide (propanil) and its metabolite (DCA) on erythroid colony formation from human cord blood progenitor cells. The IC50 values for propanil were 234.0 ± 23.7 μM for CFU-E and 441.8 ± 19.5 μM for BFU-E, whereas DCA did not reach 50% of inhibition at the doses tested except on CFU-E, which had an IC50 value of 449.9 ± 28.6 μM.

The comparison between IC50 values calculated for CFU-E, BFU-E, total erythroid colonies (TOT-E), and CFU-GM are summarized in Table 2. The IC50 values were 305.2 ± 22.6 μM (TOT-E) and >500 μM (CFU-GM) for propanil. Human erythroid progenitors were significantly more sensitive to propanil than the myeloid ones, whereas there was no significant difference in sensitivity between the two different lineages when exposed directly to DCA (IC50 values of >500 μM for both TOT-E and CFU-GM).

DISCUSSION

Pesticides can cause hematological disease and interfere with one or more hematopoietic lineages leading to neutropenia or agranulocytosis, thrombocytopenia, anemia, and, in severe cases, aplastic anemia (Fleming and Timmeny, 1993; Roberts, 1990).

As is well known in rodents, propanil and its metabolite DCA exert myelotoxic effects both in vitro and in vivo (Barnett and Gandy, 1989; Blyler et al., 1994; De Silva and Bodinayake, 1997). The selective toxicity of these two compounds with respect to erythroid or myeloid progenitors was evaluated in this study.

In vitro data on murine cells indicate that the erythrocytic lineage (CFU-E and BFU-E) is the most sensitive target for propanil and DCA, as higher concentrations of the parental compound and its metabolite were required to elicit 50% reduction in the number of CFU-GM. Both propanil and DCA reduced the number of CFU-E and BFU-E, with the latter chemical being a slightly more potent inhibitor than the parent compound.

Erythrocytes are affected by propanil in vivo, as methemoglobinemia (Singleton and Murphy, 1973) and hemolitic anemia (McMillan et al., 1991) have been reported in exposed animals and humans (De Silva and Bodinayake, 1997). Both effects are thought to be mediated by the N-hydroxylated metabolite, N-hydroxy-3,4-dichloroaniline (McMillan et al., 1990). This compound, unlike the unmetabolized parent compound, propanil, or its first immediate deacylated compound, DCA, can cause direct destruction of erythrocytes in vitro (McMillan et al., 1991). The in vitro results suggest that, at variance with mature red blood cells, either propanil or DCA directly affect bone marrow erythroid progenitor cells.

On the other hand, human progenitors were less sensitive to the toxic effects of both compounds than murine progenitors at the same concentration of exposure.

Propanil was significantly more toxic to human erythroid progenitors than to CFU-GM human progenitors as well, with the murine results underscoring the role of the heme pathway as the target of propanil toxicity in accordance with the literature and in vivo results (Vial et al., 1996).

On the other hand, DCA had only a low effect on human erythroid and CFU-GM colony development at doses tested (IC50 value for both TOT-E and CFU-GM: >500 μM), whereas the metabolite seemed to exert a higher effect than the parent compound on murine cells. These results could be explained by the interspecies differences in the metabolism of the toxicants (Gianni et al., 1990). Because the DCA target seems to be one step in the hemoglobin production pathway in rodents, this difference could also be explained by a possible diversity between murine and human enzymes involved in this pathway.

As summarized in Table 2, BFU and CFU did not show a significantly different sensitivity to DCA, whereas propanil exerted a more toxic effect on CFU-E (propanil: 234.0 ± 23.7 μM for CFU-E and 441.8 ± 19.5 μM for BFU-E). These data confirm the evidence that the two compounds investigated interfere with erythroid colony formation at different stages of the differentiation pathway and with different effects according to the dose or the time of exposure (Blyler et al., 1994). Such a selective toxicity could be explained by the action of propanil on cytokine production, mainly erythropoietin or granulocyte/macrophage colony-stimulating factor (Barnett and Gandy, 1989; Theus et al., 1993).

The range of doses tested in this work was in accordance with in vivo studies on rodents; the immunotoxic effect was seen in C57B1/6 mice when exposed intraperitoneally to doses ranging from 10 to 400 mg/kg body weight (Barnett and Gandy, 1989; Zhao et al., 1998).

The propanil doses expected to be experienced by humans are not well defined. Previous reports showed a propanil reference risk dose in humans of 0.005 mg/kg/day. In an area of rice cultivation, a propanil concentration of 0.1–0.228 μg/l in river samples and 0.0003 mg/kg in leafy vegetables was detected (Gartrell et al., 1986; Johnson et al., 1984).

In a recent study (Richards et al., 2001), the amount of propanil collected from stations in and around homes located adjacent to a rice field ranged from 3.1 to 614.7 μg/sampling surface. Our results, when compared with these data, indicate that at these ranges of concentration, propanil and its metabolite DCA do not exert a significant myelotoxic effect on human CFU-E and BFU-E progenitors.

Workers such as applicators and spotters (flaggers) for aerial applications of pesticides are usually exposed to higher pesticides concentrations. The lack of information on occupational exposure does not permit the comparison between in vitro and in vivo data. However, the results obtained at the higher concentration tested suggest further investigations to evaluate the myelotoxicity after acute and chronic exposure of workers to propanil. Furthermore, our data indicate that to avoid notable underestimation or overestimation of in vitro xenobiotic toxicity when nonhuman cell lines (e.g., murine cell lines) are employed, the use of human cell lines is fundamental for in vitro risk assessment studies.

TABLE 1

Comparison between IC50 (μM) of CFU-E, BFU-E, and CFU-GM after Direct Exposure of Murine Progenitor Cells to Test Compounds

 Propanil DCA 
Note. IC50 values (mean ± SE) were calculated by the Reed and Muench (1938) formula on at least three experiments carried out in triplicate. 
CFU-E 69 ± 12  26 ± 7 
BFU-E 18 ± 11  13 ± 5 
CFU-GM 170 ± 24 171 ± 17 
 Propanil DCA 
Note. IC50 values (mean ± SE) were calculated by the Reed and Muench (1938) formula on at least three experiments carried out in triplicate. 
CFU-E 69 ± 12  26 ± 7 
BFU-E 18 ± 11  13 ± 5 
CFU-GM 170 ± 24 171 ± 17 
TABLE 2

Comparison between IC50 (μM) of CFU-E, BFU-E, TOT-E, and CFU-GM after Direct Exposure of Human Umbilical Cord Blood Cells to Test Compounds

 Propanil DCA 
Note. TOT-E, total erythroid colonies. IC50 values (mean ± SE) were calculated by the Reed and Muench (1938) formula on at least three experiments carried out in triplicate. 
*Significantly lower (p < 0.05) than IC50 of BFU-E. 
**Significantly lower (p < 0.05p < 0.01) than IC50 of CFU-GM. 
CFU-E 234.0 ± 23.7* 449.9 ± 28.6 
BFU-E 441.8 ± 19.5 >500 
TOT-E 305.2 ± 22.6** >500 
CFU-GM >500 >500 
 Propanil DCA 
Note. TOT-E, total erythroid colonies. IC50 values (mean ± SE) were calculated by the Reed and Muench (1938) formula on at least three experiments carried out in triplicate. 
*Significantly lower (p < 0.05) than IC50 of BFU-E. 
**Significantly lower (p < 0.05p < 0.01) than IC50 of CFU-GM. 
CFU-E 234.0 ± 23.7* 449.9 ± 28.6 
BFU-E 441.8 ± 19.5 >500 
TOT-E 305.2 ± 22.6** >500 
CFU-GM >500 >500 
FIG. 1.

Concentration-dependent inhibition of BFU-E colony formation resulting from in vitro exposure of murine bone marrow cells to propanil and DCA. Data are expressed as percent vehicle colonies and represent the mean ± SE of three independent experiments performed in triplicate.

FIG. 1.

Concentration-dependent inhibition of BFU-E colony formation resulting from in vitro exposure of murine bone marrow cells to propanil and DCA. Data are expressed as percent vehicle colonies and represent the mean ± SE of three independent experiments performed in triplicate.

FIG. 2.

Concentration-dependent inhibition of CFU-E colony formation resulting from in vitro exposure of murine bone marrow cells to propanil and DCA. Data are expressed as percent vehicle colonies and represent the mean ± SE of three independent experiments performed in triplicate.

FIG. 2.

Concentration-dependent inhibition of CFU-E colony formation resulting from in vitro exposure of murine bone marrow cells to propanil and DCA. Data are expressed as percent vehicle colonies and represent the mean ± SE of three independent experiments performed in triplicate.

FIG. 3.

Concentration-dependent inhibition of CFU-GM colony formation resulting from in vitro exposure of murine bone marrow cells to propanil and DCA. Data are expressed as percent vehicle colonies and represent the mean ± SE of three independent experiments performed in triplicate.

FIG. 3.

Concentration-dependent inhibition of CFU-GM colony formation resulting from in vitro exposure of murine bone marrow cells to propanil and DCA. Data are expressed as percent vehicle colonies and represent the mean ± SE of three independent experiments performed in triplicate.

FIG. 4.

Toxic effect of propanil on CFU-E and BFU-E colony formation from human cord blood progenitors. Data are expressed as percent vehicle colonies and represent the mean ± SE. Each point represents the mean of at least three experiments.

FIG. 4.

Toxic effect of propanil on CFU-E and BFU-E colony formation from human cord blood progenitors. Data are expressed as percent vehicle colonies and represent the mean ± SE. Each point represents the mean of at least three experiments.

FIG. 5.

Toxic effect of DCA on CFU-E and BFU-E colony formation from human cord blood progenitors. Data are expressed as percent vehicle colonies and represent the mean ± SE. Each point represents the mean of at least three experiments.

FIG. 5.

Toxic effect of DCA on CFU-E and BFU-E colony formation from human cord blood progenitors. Data are expressed as percent vehicle colonies and represent the mean ± SE. Each point represents the mean of at least three experiments.

1
To whom correspondence should be addressed. Fax: 0039 0332 785336. E-mail: ilaria.malerba@jrc.it.

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,
107
–120.