Glutathione-dependent cytotoxicity of the chloroacetanilide herbicides alachlor, metolachlor, and propachlor in rat and human hepatoma-derived cultured cells

Alachlor, metolachlor, and propachlor are widely used chloroacetanilide herbicides. Their cytotoxicity in rat (Fa32) and human (Hep G2) hepatoma-derived cells was investigated, in connection with their influence on the endogenous glutathione (GSH) content, on the xenobiotic-metabolizing phase I enzymes 7-ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-depentylase (PROD), and phase II glutathione transferase (GST). The cytotoxicity was measured by the neutral red uptake inhibition assay. The following toxicity range was observed in both cell lines : propachlor>alachlor>metolachlor. When the endogenous GSH content was reduced by pretreatment of the cells with L-buthionine (S,R)-sulfoximine, the cytotoxicity of the herbicides increased strongly in both cell lines. EROD and PROD activities were dose-dependently increased to different degrees in Fa32, as was EROD in Hep G2, but no PROD activity was observed in these cells. The GSH content was not altered after 1 h treatment, and was approximately doubled after 24 h. GST activity was increased in Fa32 cells but not in Hep G2. A comparable cytotoxicity was observed for the investigated chloroacetanilides in both the rat and the human cell lines. Different interactions with xenobiotic-metabolizing phase I and II enzymes were observed, and GSH showed a protective effect against the acetanilides in both cell lines.     

Herbicide Induced Oxidative Stress in Lettuce, Beans, Pea Seeds and Leaves

The effects of different paraquat, alachlor and metolachlor concentrations on superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX) activities and pigment contents in lettuce, bean and pea seeds and leaves were studied. Under paraquat (1.0 and 2.0 M) treatment declined SOD and CAT activities were observed in seeds and undetectable ativity of GPX. Germination of all investigated seeds was completely inhibited. All used concentrations of alachlor and metolachlor inhibited antioxidant enzyme activities in seeds but did not prevent germination and growth. In leaves, lower concentrations of these herbicides increased activities of antioxidant enzymes but at the highest herbicide concentrations (200 M) activities of investigated enzymes declined. The pigment contents the leaves decreased due to alachlor and metolachlor treatment in a concentration dependent manner.     

Effects of Eight Herbicides on In Vitro Hatching of Heterodera glycines

Laboratory studies were conducted to evaluate effects of selected herbicides on hatching of free eggs of the soybean cyst nematode, Heterodera glycines. The herbicides used were Atrazine (atrazine), Basagran (bentazon), Bladex (cyanazine), Blazer (acifluorfen), Command (clomazone), Lasso (alachlor), Sonalan (ethalfluralin), and Treflan (trifluralin). Treatments comprised two concentrations of commercial herbicide formulations and deionized water and 3.14 mM zinc sulfate as negative and positive controls, respectively. Eggs were extracted from females and cysts, surface disinfested, and incubated in herbicide or control solutions at 25 ± 2 C in darkness. Hatched second-stage juveniles were counted every other day for 24 days. Hatching of H. glycines eggs in 50 and 500 μg/ml Blazer was 42 to 67% less than that in deionized water and 6l to 78% less than that in zinc sulfate solution. Zinc sulfate significantly increased hatching activity in 50 μg/ml but not 500 μg/ml Blazer. The other herbicides tested at various concentrations had no significant effect on egg hatching. The specific component of Blazer inhibiting egg hatching is unknown. Suppression of hatching by Blazer indicates that this postemergence soybean herbicide may have a potential role in managing H. glycines.     

Development of Heterodera glycines as Affected by Alachlor and Fenamiphos

A series of greenhouse experiments was conducted to elucidate the postinfection development of Heterodera glycines in response to applications of alachlor and fenamiphos. The rate of H. glycines maturation on a susceptible soybean cultivar was not altered by 1.0 μg alachlor/g soil but was completely inhibited by 1.0 or 1.5 μg fenamiphos/g soil. An alachlor-fenamiphos combination allowed development after an initial 300-degree-day delay. Nematode maturation on the resistant soybean cultivar Centennial with 1.0 μg alachlor/g soil was similar to that observed on an untreated resistant control. Twice as many females matured on Centennial plants growing in alachlor-treated soil as on untreated Centennial plants. Fenamiphos in combination with alachlor (1.0 μg a.i./g soil) allowed development on Centennial at half the rate of the resistant control. This antagonism between alachlor and fenamiphos on development may help to explain late season population resurgence of H. glycines observed with field application of these pesticides.     

Effect of Planting Date,Alachlor, and Fenamiphos on Heterodera glycines Development

The effects of alachlor (2.25 kg a.i./ha) and fenamiphos (2.25 kg a.i./ha) on the penetration and development of Heterodera glycines were examined on Glycine max cultivars Deltapine 105 planted 29 April, 29 May, and 29 June 1986 and Deltapine 105 and Centennial planted 15 May, 15 June, and 15 July 1987. Penetration was lowest on the third planting of soybeans and on fenamiphos-treated plants. Development from second-stage juveniles to adult females required 270 (1986) and 260 (1987) DD20/32 on roots from the first planting control and alachlor treatments. Fenamiphos, alone or with alachlor, retarded development in Deltapine 105 (1986) and in Centennial (1987). Males matured in roots from the second planting in 190 (1986) and 180 (1987) DD20/32 regardless of treatment or cultivar. No development occurred in roots from the third planting until 400 DD20/32 in 1986, but in 1987 development was similar to that in roots from the second planting. Nematode development was similar in alachlor-treated and control roots regardless of planting date. Fenamiphos restricted nematode penetration on most planting dates and slowed development. Simultaneous applications of alachlor and fenamiphos usually also inhibited development.     

Effects of inhibitors on the plasma membrane and mitochondrial adenosine triphosphatases of Neurospora crassa

A comparative study has been made of the effects of a variety of inhibitors on the plasma membrane ATPase and mitochondrial ATPase of Neurospora crassa. The most specific inhibitors proved to be vanadate and diethylstilbestrol for the plasma membrane ATPase and azide, oligomycin, venturicidin, and leucinostatin for mitochondrial ATPase. $\text{N,N′-}\text{Dicyclohexylcarbodiimide}$, octylguanidine, triphenylsulfonium chloride, and quercetin and related bioflavonoids inhibited both enzymes, although with different concentration dependences. Other compounds that were tested (phaseolin, fusicoccin, deoxycorticosterone, alachlor, salicyclic acid, $\text{N-1-}\text{napthylphthalamate}$, triiodobenzoic acid, cyclic AMP, cyclic GMP, theobromine, theophylline, and histamine) had no significant effect on either enzyme. Overall, the results indicate that the plasma membrane and mitochondrial ATPases are distinct enzymes, in spite of the fact that they may play related roles in H⁺ transport across their respective membranes.     

Characterization of Cytosolic Glutathione S-Transferase in Spruce Needles

Active glutathione S-transferase (GST) has been purified from needles of Norway spruce (Picea abies L. Karst.). Two isoforms of the enzyme which exhibit different physico-chemical and catalytic properties were separated by (NH₄)₂SO₄ fractionation, affinity chromatography on epoxy-activated 4% cross-linked beaded agarose, using glutathione as the ligand, ion-exchange chromatography, and isoelectric focusing. The isozymes have pI values of 5.5 (GST I) and 4.3 (GST II). Both GST isozymes are homodimeric proteins with subunit sizes of 26 kD (GST I), and 23 kD (GST II). The kinetic properties of the enzymes are described and compared with other plants GSTs. Only GST II is able to conjugate the pesticides fluorodifen and alachlor.     

Effects of humic acid-herbicide interactions on the growth of Pisum sativum in nutrient solution

Slight increases in root length and dry matter production were measured in roots and shoots of Pisum sativum seedlings, when germinated seeds were grown for eight days in a controlled environment chamber, on Nitch nutrient solution to which 10 mg L^-1 soil humic acid (HA) had been added. A concentration of 100 mg L^-1 of HA produced a small reduction in dry matter production of shoots. In the presence of herbicides, 10 mg L^-1 cycluron or 100 mg L^-1 prometone, root elongation and shoot growth was reduced by varying degrees with respect to the control. Nutrient solutions containing 100 mg L^-1 cycluron, 10 or 100 mg L^-1 alachlor, or as little as 0.1 mg L^-1 of 2,4-D resulted in an almost complete suppression of the growth of pea seedlings. The addition of 10 or 100 mg L^-1 of HA to nutrient solution containing the herbicide was found to be either ineffective in enhancing growth, or even further reduced growth, compared to samples grown in the presence of the herbicide alone. The results of this preliminary study suggest that the generally accepted view that humic substances exert a positive effect on plant growth may not occur when humic substances interact with herbicides.     

Population Dynamics of Heterodera glycines and Soybean Response in Soils Treated with Selected Nematicides and Herbicides

Two field experiments were conducted in two locations to determine the effects of the nematicides aldicarb, phenamiphos, and ethoprop and/or the herbicides alachlor, linuron, or metribuzin on the population dynamics of Heterodera glycines and soybean growth and yield. Population densities of H. glycines were greater, at some time during the growing season, in several treatments with alachlor alone and in combination with nematicides. Numbers of H. glycines at harvest were greater in plots treated with aldicarb than in those treated with ethoprop or phenamiphos. The numbers in aldicarb treated plots were generally reduced when plots also received a herbicide. Soybean yields were negatively correlated with numbers of H. glycines eggs and juveniles in early to mid season but positively correlated with late season population densities.