The effect of diflubenzuron [1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea] on soybean [Glycine max (L.) Merr.] photosynthesis,respiration, and leaf ultrastructure

The effect of the insecticide diflubenzuron [1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea] on photosynthesis, respiration, and leaf ultrastructure of soybean [Glycine max (L.) Merr., cv. Swift] was examined on plants treated at the second trifoliate leaf stage with 0, 0.067, and 0.269 kg of active ingredien/ha of diflubenzuron. Photosynthesis and respiration were measured with an infrared CO₂ analyzer in an open flow system prior to diflubenzuron application and at 4, 24, 48, and 96 hr after treatment with diflubenzuron. Diflubenzuron had no effect on soybean photosynthesis at any rate examined. Respiration was stimulated by the high rate (0.269 kg/ha) in a transitory manner. Tissue samples removed from both old and new leaves, 9 days after diflubenzuron application, were used for the ultrastructure study with the transmission electron microscope. The lower trifoliate leaves contained more starch grains than the upper ones being formed after treatment, but no aberrations or degradation of leaf ultrastructure due to diflubenzuron treatment were evident.     

In vitro activity and binding characteristics of the hydroxybenzonitriles in chloroplasts isolated from Matricaria inodora and Viola arvensis

The electron transport inhibition, uncoupling, and binding of ioxynil and bromoxynil salts is compared in chloroplast fragments isolated from two weed species with contrasting responses to the hydroxybenzonitriles. Ioxynil Na was three to four times more inhibitory than bromoxynil K towards DCPIP and SiMo reduction in both Matricaria inodora and Viola arvensis. Ioxynil Na was also a more potent uncoupler of PSI-dependent electron transport from ascorbate/DCPIP to methyl viologen. Uncoupling occurred at concentrations higher than those that inhibited electron transport. Binding studies with [¹⁴C]bromoxynil K and [¹⁴C]ioxynil Na salts revealed slightly biphasic curves with no significant difference in the amounts of the two herbicides bound at a given concentration. The ratios of inhibition constant (K_i) and binding constant (K_b) were approximately one for ioxynil Na and three for bromoxynil K. Radiolabelled herbicide displacement studies revealed that ioxynil Na could partially displace bound [¹⁴C]bromoxynil K, but bromoxynil K could not displace bound ioxynil Na at biochemically active concentrations. Ioxynil Na may be a more effective inhibitor than bromoxynil K because it binds more strongly to the thylakoid membrane.     

The action of formamidines on octopamine receptors in the locust

The actions of a range of formamidines have been investigated biochemically and physiologically on octopamine receptors in the locust Schistocerca gregaria. All the formamidines tested [chlordimeform (CDM), demethylchlordimeform (DCDM), amitraz, and UK 16353] mimicked the action of octopamine by (1) increasing the amplitude and relaxation rate of slow motorneurone tension in the extensor-tibiae muscle of the hind leg and (2) changing the levels of cyclic AMP in this muscle. UK 16353 was most effective in changing these parameters, followed by DCDM then amitraz and CDM. The formamidine-induced increase in cyclic AMP levels was reduced or completely blocked by phentolamine, an antagonist of insect octopamine receptors. The time course for the increase in cyclic AMP was followed for 30 min by incubating muscles in 10^?5M DCDM. The increase was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. In the presence of this compound, the response peaked within 5 min, before declining to a lower plateau after 12 min. The response to 10^?5M CDM was lower and the maximum increase occurred after 7 min, then rapidly declined. Both formamidines increased cyclic AMP in a dose-dependent way with a threshold of between 5 × 10^?7 and 10^?8M. The results are discused in terms of the relationship between the biochemical and physiological actions of the formamidines in this preparation.     

Rates of sulfide oxidation in cotton,carrot, and tobacco cultured plant cells measured with a model aromatic alkyl-sulfide

p-Chlorophenylmethylsulfide (PCPMS) was enzymatically sulfoxidized to p-chlorophenylmethylsulfoxide (PCPMSO) in aerobic cotton, carrot, and tobacco cell suspension cultures. Neither boiled nor freeze-killed cell cultures were competent to sulfoxidize PCPMS to PCPMSO. The sulfone, p-chlorophenylmethylsulfone (PCPMSO₂), was not produced in any of the three species. The rates of PCPMS sulfoxidation, were cotton > carrot > tobacco. The apparent K_m for carrot cells was 88 μM PCPMS (14 μg/ml), while the apparent V_max was 7 nmol PCPMSO/mg whole cell protein/hr. These rates of sulfide oxidation are higher than previously reported in intact plants.     

Differential inhibition responses caused by DDT on oligomycin-sensitive Mg2+-ATPase activity: Nature of the requirements for DDT sensitivity

Earlier communications from this laboratory have shown that DDT inhibited oligomycin-sensitive Mg²⁺-ATPase (EC 3.6.1.3) but that its active component, F₁, was not affected. In the present investigation evidence has been obtained to determine the nature of the requirements for DDT sensitivity. The results showed that DDT sensitivity was conferred to F₁ from pig heart mitochondrial preparations when it was bound to F₀ from the same preparation. The F₁ from house fly (Musca domestica L) thorax was able to bind to F₀ from pig heart. This combination showed similar sensitivity to that of the original F₁-F₀ combination from pig heart mitochondria. However, when F₁ from pig heart mitochondria was incorporated into F₀ depleted in oligomycin sensitivity-conferring protein (OSCP) from the same source, the resulting ATPase activity was insensitive to DDT. Addition of crude (50–200 μg) or purified (5–20 μg) OSCP in the above preparation restored DDT sensitivity. Presence of dioleyl or dipalmitoyl phosphatidyl choline or Triton X-100 in the reaction medium antagonized the DDT inhibitions. Depletion of phospholipids from submitochondrial membrane preparations (SMP) decreased ATPase activity. Addition of dioleyl or soybean phosphatidyl choline to this lipid-depleted preparation restored DDT sensitivity. Evidence presented suggests that DDT acted on F₁ in association with one or more membrane components and that OSCP and phospholipid were essential for DDT sensitivity.     

Uptake of dieldrin,lindane, and DDT by isolated rat hepatocytes

Studies of the uptake of dieldrin, lindane, and DDT by isolated rat hepatocytes in Krebs-Henseleit salt solution containing 2.34% bovine serum albumin have shown that the partition equilibrium of these hydrophobic organochlorine compounds is established very rapidly between the hepatocytes and the extracellular medium by reversible uptake and release processes. In the hepatocyte suspension system, these compounds are in dynamic partition equilibrium between the hepatocytes and the Krebs-Henseleit salt solution, and also between the bovine serum albumin molecules and the solution. The respective rate constants for the uptake and release processes were 0.83 and 2.52 × 10^?2 sec^?1 in a 2-ml suspension containing 10⁶ cells. It has thus been demonstrated that the transport of hydrophobic organochlorine compounds between the hepatocytes and the extracellular medium is a much faster process than the metabolic transformation reaction in hepatocytes.     

Cytochrome P-450 content and aldrin epoxidation to dieldrin in isolated rat hepatocytes

A rat hepatocyte suspension effectively epoxidized aldrin to dieldrin with a V_max of 7.19 mol/mol P-450/min and a K_m of 9.27 μM. Viability and metabolic activity were stable for 6 hr after isolation when cells were maintained at room temperature (20°C) with the gentle introduction of $\text{O}{}_2\text{CO}{}_2$ onto the surface of the suspension. The cytochrome P-450 content of the suspension was 303 pmol/10⁶ cells. Primary maintenance culture of the cells also epoxidized aldrin. During culture for 3 days, metabolic activity decreased slowly day by day. Metabolic activity of microsomal fraction from rat liver was also examined. Microsomes epoxidized aldrin with a V_max of 5.11 mol/mol P-450/min and a K_m of 1.64 μM. Significant loss of some subspecies of cytochrome P-450 during fractionation of liver homogenate was indicated.     

Hepatic disposition of parathion: Uptake by isolated hepatocytes and chromatographic translobular migration

Isolated rat hepatocytes suspended in Waymouth's medium absorbed parathion rapidly and reversibly until the intracellular parathion concentration reached more than 300 times the ambient concentration. The distribution quotient q, defined as the ratio of intra- and extracellular concentrations at equilibrium, decreased when horse serum was added to the medium. The high hepatocyte affinity and rapid uptake of parathion suggested that this compound might be almost completely absorbed by periportal hepatocytes in the perfused liver and migrate downstream chromatographically through the lobule. Parathion infusion experiments verified this prediction and showed that the migration rate is dependent on the q value. The chromatographic feature may be useful as a basis for nonhistological investigation of intralobular hepatocyte heterogeneity. The lobule may function as a reverse-phase chromatograph also for many other unionized xenobiotics. Implications of the findings in the hepatic disposition of xenobiotics in vivo are discussed.     

Herbicidal interference with the photochemical activities of chloroplasts (in vivo and in vitro) of certain crop and weed species

The independent modes of action of diuron and atrazine on the photochemical activities of chloroplasts (In vivo and in vitro) from the leaves of crop plants Pisum sativum and Pennisetum typhoides and the weeds Amaranthus viridis and Cyperus rotundus were investigated. Hill reaction activity (DCPIP photoreduction) of in vivo chloroplasts (chloroplasts isolated from herbicide-sprayed plants) was unaffected by treatment at sublethal or intermediate levels of diuron or atrazine while that of in vitro chloroplasts (chloroplasts incubated in the required herbicidal concentration) was severely inhibited. The ferricyanide catalyzed noncyclic photophosphorylation was markedly reduced in both the in vivo and in vitro chloroplast systems. N-Methyl phenozonium sulfate (PMS)-mediated cyclic photophosphorylation was inhibited in the in vivo system while a pronounced enhancement of activity was noticed in the in vitro chloroplasts. The rate of NADP⁺ photoreduction was severely inhibited in the in vitro chloroplasts. The unaffected in the in vivo system. The herbicidal effects on the photoreactions of isolated chloroplasts were compared with chloroplasts isolated from herbicide-sprayed plants.     

Influence of pyrethroid ester,oxime ether,and other central linkages on insecticidal activity,hydrolytic detoxification,and physicochemical parameters

The relative potency to target and nontarget insects of ester pyrethroids and the analogous oxime ethers, and the degree of synergism of the esters with tributyl phosphorotrithioate, provide a useful guide to the importance of esterase detoxification in species specificity. These criteria indicate that esterase detoxification is a more important component of pyrethroid tolerance in Chrysopa carnea and Cryptolaemus montrouzieri larvae than in Exochomus flavipis larvae and Musca domestica adults. Studies with 3-phenoxybenzyl 2-(4-chlorophenyl)-2-cyclopropylacetate and the corresponding 2,3,4,5,6-pentafluorobenzyl ester, their oxime ether analogs, and permethrin and its thiol ester and amide analogs provide evidence that the high insecticidal activity of some ester and E-oxime ether pyrethroids, relative to that of the corresponding thiol ester and amide, is more closely associated with the dipole moment and polarizability of the central linkage and its resistance to esterase detoxification than with bond lengths or lipophilicities conferred by a specific linkage. Pentafluorobenzyl tetramethylcyclopropanecarboxylate is very effective in the vapor state for house fly control.     

Deuterium isotope effects on the formation of mercapturic acids from lindane in rats

Metabolism experiments with rats showed that significant isotope effects ($\text{k}{}_{\mathrm{<mtext>H</mtext>}}\text{k}{}_{\mathrm{<mtext>D</mtext>}}\text{= 2.4}\text{to}\text{3.5}$) were associated with the in vivo formation of dichloro and trichlorophenylmercapturic acids from a 1:1 mixture of normal and hexadeuterated lindane. This is evidence that rate-determining dehydrogenation and dehydrochlorination, both of which proceed with significant isotope effects, are essential in the pathway of dichloro- and trichlorophenylmercapturic acid formation from lindane. No significant primary isotope effects were associated ($\text{k}{}_{\mathrm{<mtext>H</mtext>}}\text{k}{}_{\mathrm{<mtext>D</mtext>}}\text{= 1.31 ± 0.17}$) with the formation of monochlorophenylmercapturic acid. This suggests that the 1,2-dechlorination to tetrachlorocyclohexene followed by glutathione conjugation is the probable pathway that produces this metabolite from lindane.     

Increased cyclic nucleotides in several tissues of the cockroach and mouse following treatment with toxaphene

Cylic AMP (cAMP) and cyclic GMP (cGMP) concentrations in toxaphene-treated cockroaches, Leucophaea maderae (Fab.), increased in all tissues sampled, with the greatest increases occurring at 24 hr for cAMP and 168 hr for cGMP. Asymptomatic mice treated with 112 mg/kg of toxaphene were not significantly different from controls. Symptomatic mice showed increased cAMP in all tissues examined and an increase in testicle cGMP. The greatest increases in cAMP occurred during the more advanced stages of toxaphene poisoning.     

Differential effects of phenoxy herbicides on light-dependent 14CO2 fixation in isolated cells of C3 and C4 plants

Cells were isolated from the developing leaves of Ipomoea aquatica and Digitaria sanguinalis. The effects of phenoxy alkanoic acid herbicides on light-dependent ¹⁴CO₂ fixation and oxygen evolution in these leaf cells were studied. (2,4-Dichlorophenoxy)acetic acid and (2,4,5-trichlorophenoxy) acetic acid (2,4,5-T and 2,4-D) caused a 20% stimulation of ¹⁴CO₂ fixation at 0.8 × 10^?5M and an inhibition at 1 × 10^?4M in I. aquatica leaf cells. Temperature seemed to have a marked influence on such action. No effect or very little effect was observed in the leaf cells of D. sanguinalis. The nonactive (2,4,6-Trichlorophenoxy)acetic acid (2,4,6-T) caused a similar stimulation of CO₂ fixation as 2,4-D and 2,4,5-T at low concentrations in I. aquatica leaf cells, but no inhibition was observed at high concentration. Increase of hight intensity increased the rate of CO₂ fixation in both control and 2,4,6-T-treated cells; however, the percentage of stimulation remained the same. At stimulatory concentration, all three compounds did not cause any stimulation in either photosystem I and II or photosystem II-mediated oxygen evolution. At higher concentrations, the differential effects of 2,4-D and 2,4,5-T on the light-induced CO₂ fixation and photosystem II-mediated oxygen evolution in the I. aquatica leaf cells and D. sanguinalis mesophyll (ms) cells may be attributed in part to their selective action against dicotyledonous plants.     

The mechanism of resistance to lindane and hexadeuterated lindane in the Third Yumenoshima strain of house fly

The factors which cause lindane resistance in the Third Yumenoshima strain, a strain of house flies highly resistant to insecticides, were studied using hexadeuterated lindane. Hexadeuterated lindane has the same physicochemical properties as lindane, but the former is much less biodegradable than the latter. The LD₅₀ ratio of lindane to hexadeuterated lindane in this strain, deuterium isotope effect on LD₅₀ values, was larger than that in S_NAIDM, a susceptible (nonresistant) strain. The penetration rates of labeled and nonlabeled lindane through the insect cuticle were about the same for both strains. Thus, penetration rate does not cause resistance. The metabolic degradation of lindane in the resistant strain in vivo occurred much faster than in the susceptible strain. This was also the case for lindane degradation processes in vitro such as microsomal oxidation and glutathione conjugation. In both strains, significant isotope effects were observed in the degradation rates in vitro of labeled and nonlabeled lindane. Therefore, principal biodegradation and detoxication pathways should include reactions which cleave the CH bonds. When the much less biodegradable d₆ counterpart of lindane was applied to both strains, the susceptible strain became much more highly intoxicated than the other within 20 to 30 min. This indicates that a combination of both greater degradability and probably lower sensitivity at the action site are the main factors underlying resistance in the Third Yumenoshima strain.     

In vitro effects of DDT analogs on trout brain Mg2+-ATPases: I. Specificity and physiological significance

A continuous steady-state assay procedure was used to investigate the effects of DDT and several analogs on the in vitro Mg²⁺-stimulated adenosinetriphosphatase of a trout brain mitochondrial fraction. Pharmacological dissection of the enzyme with oligomycin, dicyclohexyl-carbodiimide, and azide failed to yield a fraction specifically sensitive to the organochlorines. At 25°C, low doses of DDT (≤1.35 μmol/mg of protein) stimulated enzyme activity, while methoxychlor was stimulatory at all doses. Higher doses of DDT and of several analogs caused only 45.5% or less inhibition at 25°C, but inhibition increased at lower temperatures. The physiological significance of these effects is discussed.     

Effects of herbicides and herbicide analogs on [14C]leucine incorporation by suspension-cultured Solanum nigrum cells

Assays of [¹⁴C]leucine incorporation were used to measure effects of herbicides on suspensioncultured heterotrophic Solanum nigrum cells. Most herbicidal vs. nonherbicidal chemicals in a set of 47 compounds could be distinguished from each other based on their extent of inhibition of leucine incorporation by S. nigrum cells. Herbicides which failed to inhibit leucine incorporation were photosynthetic inhibitors. Both phytotoxic and nonphytotoxic thiocarbamate analogs (as determined by whole-plant studies) tended to inhibit leucine incorporation. It was concluded that the leucine incorporation screen could detect a majority of compounds tested which are herbicidal, and that it may also be useful to detect compounds which have cellular toxicity which is not observed in the whole plant.     

In vitro effects of malathion and O,O,S-trimethyl phosphorothioate on cytotoxic T-lymphocyte responses

Oral administration of O,O,S-trimethyl phosphorothioate (OOS), an impurity present in technical formulations of malathion, has been shown to be associated with a high incidence of pneumonia in rats and to be highly immunosuppressive in mice. Based on these findings, an in vitro model was established to study the effect of this and other organophosphorus compounds on murine cytotoxic T-lymphocyte (CTL) responses. The organophosphorus compounds were tested for their ability to block in vitro generation of CTL responses to alloantigen and/or the expression of these cytotoxic responses. Responses were generated in C57Bl/6 (H-2^b) spleen cells to mitomycin C-blocked P815 (H-2^d) tumor cells. The cytotoxicity of the cultured splenocytes to P815 target was measured using a 4-hr chromium release assay. These data demonstrated that malathion was able to block the ability of splenocytes to sensitize to P815 at concentrations as low as 25 μg/ml, but was not able to block the expression of cytotoxicity by mature killer T cells. The same was true for OOS which had been activated by preincubation with rat liver postmitochondrial supernatant (PMS). Activated OOS blocked the generation of CTL responses at concentrations as low as 75 μg/ml while having no effect on mature cytotoxic cells. In fact, both malathion and activated OOS were no longer able to suppress CTL responses if treatment was performed as early as 24 hr after exposure to antigen. Additionally, it was demonstrated that when malathion was preincubated with PMS it was no longer suppressive and that OOS without activation failed to suppress CTL responses.     

O,O,S-Trimethyl phosphorothioate effects on immunocompetence

O,O,S-Trimethyl phosphorothioate (OOS), a contaminant of technical formulations of some organophosphorus pesticides, was found to be immunotoxic at subtoxic doses in female C57Bl/6 mice. Mice treated orally with acute doses of 10 mg/kg OOS show no overt toxic signs such as weight loss or malaise. In addition, the levels of serum cholinesterase was not decreased. Histopathologic investigation demonstrated no alterations in liver, lung, kidney, heart, skin, brain, spleen, or gut. The LD₅₀ for delayed toxicity was approximately 35 mg/kg. Despite the lack of general toxic changes at doses of 5–10 mg/kg OOS, specific immunotoxic changes were found. The humoral or cell-mediated immune response of splenocytes from mice treated with 10 mg/kg OOS to in vivo immunization was diminished with respect to control animals. Responses were measured in ex vivo assays. Cytotoxic T-lymphocyte (CTL) responses were assessed by alloimmunization with the tumor P815 followed by a ⁵¹Cr release assay done ex vivo with splenic lymphocytes. Humoral responses were assessed by immunization with sheep red blood cells followed by a Jerne plaque assay to determine anti-sheep red blood cell antibody. Both cellular and humoral responses could be stimulated in vitro using cells from OOS-pretreated, primed animals, thus indicating that no permanent cellular elterations had occurred.     

The mode of action and neurotoxicity of mirex,chlordecone, and four hydrogenated mirex analogs

The toxicity and neurological effects of mirex, chlordecone, and four hydrogenated mirex analogs were evaluated on the American cockroach. The severity of poisoning symptoms correlated with the ability of each compound to increase spontaneous activity and prolong synaptic afterdischarge in ganglia of the ventral nerve cord. Afterdischarge across the metathoracic ganglion was consistent with a characteristic wing splaying symptom in mirex-poisoned cockroaches. The actions of hemicholinium-3 and nicotine on nerve cords from mirex-poisoned cockroaches are described and are consistent with a hypothesis that the increased spontaneous activity and afterdischarge are the result of enhanced transmitter release in ganglia of poisoned animals.