Effect of piperonyl butoxide and diphenylamine on lipid peroxidation in ozonated chloroplasts

Lipid peroxide (LPO) formation was remarkable when isolatedtobacco chloroplasts were bubbled with high concentrations ofozone, though the fatty acid composition and the fractionationpattern of glycolipids and phospholipids in the chloroplastlipids changed little after ozone fumigation of the leaves.Piperonyl butoxide (PB), a potent protectant against ozone injury,strongly inhibited LPO formation in ozonated chloroplasts. PBalso prevented ozone-induced decreases in the amounts of linolenicand linoleic acids in the chloroplast lipids. These resultssuggest that PB inhibition of LPO formation may be involvedin the protective mechanism against ozone phytotoxicity. However,the mode of PB action differed on some points from that of diphenylamine,which is an antioxidant and also effective against ozone injury.The mode of PB action is discussed. ¹ Present address: The Central Research Institute, Japan Tobacco& Salt Public Corporation, Umegaoka, Midori-ku, Yokohama227, Japan. (Received July 5, 1976; )     

Preventive effect of piperonyl butoxide on cyclophosphamide-induced teratogenesis in rats

BACKGROUND: Cyclophosphamide induces fetal defects through metabolic activation by cytochrome P-450 monooxygenases (CYP). The effects of piperonyl butoxide (PBO), a CYP inhibitor, on the fetal development and external, visceral, and skeletal abnormalities induced by cyclophosphamide were investigated in rats. METHODS: Pregnant rats were daily administered PBO (400 mg/kg) by gavage for 7 days (the 6th to 12th day of gestation), and intraperitoneally administered with cyclophosphamide (12 mg/kg) 4 h after the final treatment. On the 20th day of gestation, maternal and fetal abnormalities were determined by Cesarean section. RESULTS: Cyclophosphamide reduced fetal body weights by 30–40% without increasing resorption or death. In addition, it induced malformations in live fetuses: 100, 98, and 98.2% of the external (head and limb defects), visceral (cerebroventricular dilatation, cleft palate, and renal pelvic/ureteric dilatation), and skeletal (acrania, vertebral/costal malformations, and delayed ossification) abnormalities, respectively. The pre-treatment of PBO greatly decreased mRNA expression and activity of hepatic CYP2B, which metabolizes cyclophosphamide into teratogenic acrolein and cytotoxic phosphoramide mustard. Moreover, PBO remarkably attenuated cyclophosphamide-induced body weight loss and abnormalities of fetuses; score 3.57 versus 1.87 for exencephaly, 75.5% versus 42.5% for limb defects, 65.3% versus 22% for cerebroventricular dilatation, 59.2% versus 5.1% for cleft palate, score 1.28 versus 0.93 for renal pelvic/ureteric dilatation, 71.9–82.5% versus 23–45.9% for vertebral/costal malformations, and 84.2% versus 57.4% for delayed ossification in cyclophosphamide alone and PBO co-administration groups. CONCLUSIONS: These results suggest that repeated treatment with PBO may improve cyclophosphamide-induced body weight loss and malformations of fetuses by down-regulating CYP2B. Birth Defects Res (Part B) 86:402–408, 2009. © 2009 Wiley-Liss, Inc.     

Effect of ozone on photosystem II of tobacco chloroplasts in the presence of piperonyl butoxide

Inhibitors of photosynthetic electron flow and electron donorsfor photosystem II effectively protected tobacco leaves againstozone injury, suggesting that this photosystem is closely linkedwith the development of ozone injury. Hill reaction activityof the chloroplasts isolated from tobacco leaves immediatelyafter ozone fumigation was significantly lower than that ofthe chloroplasts isolated from non-fumigated leaves. This ozone-induceddecrease in Hill reaction activity was partially prevented whenthe leaves were pretreated with piperonyl butoxide (PB), whichis highly effective against ozone injury. Ozone bubbling into the isolated chloroplast suspension markedlydecreased the rate of DPIP photoreduction. PB reduced this decreaseabout 50%. PB also prevented the ozone-induced decrease of chlorophylland carotenoids. ¹Present address: The Central Research Institute, Japan Tobacco& Salt Public Corporation, Umegaoka, Midori-ku, Yokohama227, Japan (Received June 14, 1976; )     

Effect of staphylococcal alpha-hemolysin upon anion transport in the rabbit erythrocyte

Equilibrium exchange of SO₄^2? was measured prior to and during hemolysis in rabbit erythrocytes exposed to staphylococcal α-hemolysin. The anion-transport protein of the rabbit erythrocyte has also been identified. Equilibrium exchange of SO₄^2? was measured by both efflux and influx of ³⁵SO₄^2?. The rate of influx of SO₄^2? in rabbit erythrocytes exposed to α-hemolysin was twice that of the untreated cells. The rate of SO₄^2? efflux was unchanged by α-hemolysin. Inhibition of anion exchange with 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) did not inhibit hemolysis, therefore, the increased influx of SO₄^2? may occur through a DIDS-insensitive pathway.     

Renal function and organic anion and cation transport during dehydration and/or food restriction in chickens

The effect of dehydration in the presence or absence of continued food intake on renal function was evaluated in chickens. In addition, renal transport of organic anions and cations under these conditions was assessed in vitro by uptake of ¹⁴C-para-aminohippuric acid and ¹⁴C-tetraethylammonium bromide by renal slices. Water restriction with continued food intake resulted in increases in serum osmolality and serum concentrations of sodium, uric acid, calcium and total protein. If food was restricted in addition to water, only serum osmolality and sodium concentration were significantly increased after 48 hours. Dehydration with continued access to food resulted in marked decreases in extracellular fluid volume, glomerular filtration rate and effective renal plasma flow. If food was restricted during dehydration, the decrease in effective renal plasma flow was attenuated despite reductions in glomerular filtration rate and extracellular fluid comparable to that seen in dehydrated birds allowed free access to food. Transport of organic anions was significantly increased after 24 and 48 hours of water restriction, regardless of whether food was withheld. Enhanced transport of organic anions in the presence of decreased glomerular filtration rate and effective renal plasma flow during dehydration may promote precipitation of urates and nephrosis in chickens.Abbreviations cpm counts per minute - dpm disintegrations per minute - ECF extracellular fluid - ERPF effective renal plasma flow - GFR glomerular filtration rate - PAH para-aminohippuric acid - SEM standard error of the mean - TEA tetraethylammonium bromide     

Effect of cell age and phenylhydrazine on the cation transport properties of rabbit erythrocytes

We studied the effect of cell age on the cation transport systems of rabbit erythrocytes by increasing the proportion of circulating young erythrocytes with either repeated bleeding or with phenylhydrazine (PHZ) treatment. We found that when the reticulocyte content of rabbit blood is increased by bleeding (from 1 to 40–50% of the circulating red cells), the response of the various transport pathways differs. The largest increase (fivefold) was found in the activity of K-CI contransport which peaked 3 days after the last bleeding. The Na-K pump activity peaked at a similar time, but the % increase was twofold less than the K-CI contransport. There was very small increase in the activity of the Na-Li exchange, whereas the Na-H exchange reached peak values 10 days after the last bleeding (twofold increase), when activities of K-Cl contransport and Na-K pump had returned to almost normal levels. In vivo PHZ treatment resulted in anemia and marked reticulocytosis (80–90% of circulating cells). Transport rates were markedly increased (Na-K pump 9.6-fold, Na-H exchange 6.8-fold, Na-Li exchange 2.75-fold; K-CI contransport: 10–20-fold). When blood from PHZ-treated rabbits was incubated in vitro for 24–48 hour, red cell volume and K content decreased. This process was associated with a 70% reduction in the activity of the K-CI contransport after 24 hours and a 90% reduction after 48 hours. The activity of the other systems also declined and approached baseline values after 48 hours. Loss of transport activity was not affected by 10 μM E-64, whereas 10 mM methylamine reduced the inactivation of the Na-H exchange and of the Na-Li exchange. PHZ treatment of rabbit red cells in vitro resulted in marked increase of the K-CI contransport and inhibition of Na-K pump, Na-H exchange, and Na-Li exchange. These effects were abolished by DTT, with the exception of the Na-K pump inhibition, which was DTT insensitive. Thus both cell age and oxidative damage are important determinants of cation transport in rabbit red cells. © 1993 Wiley-Liss, Inc.     

Effect of arachidonic acid and the chemotactic factor F-Met-Leu-Phe on cation transport in rabbit neutrophils

A detailed examination of the effects of exogenous arachidonate on cation metabolism in rabbit neutrophils was undertaken. Arachidonic acid stimulates the movement of ⁴⁵Ca into and out of the neutrophils with a net result, in the presence of extracellular calcium, of increasing the steady-state level of ⁴⁵Ca. Arachidonate also increases the uptake of ²²Na. These effects of arachidonate are specific to these cations, concentration-dependent, and sensitive to lipoxygenase inhibitors. At the concentrations used in this study arachidonate does not influence the permeability of human erythrocytes to ⁴⁵Ca. Furthermore, both arachidonic acid and F-Met-Leu-Phe release calcium from a previously unexchangeable intracellular pool and the effect of the two stimuli are not additive. Arachidonic acid-dependent, but not F-Met-Leu-Phe-dependent, calcium release is sensitive to lipoxygenase inhibitors. These two stimuli thus appear to release is sensitive to lipoxygenase inhibitors. These two stimuli thus appear to release calcium from the same pool(s) by separate mechanisms. The results summarized above are consistent with the hypothesis that one or more arachidonate metabolites are involved in the mechanism underlying the chemotactic factor induced permeability changes in rabbit neutrophils.     

The water and cation content of nonmetabolizing perfused rabbit kidneys.

Rabbit kidneys treated with cyanide and iodoacetate have been perfused with solutions containing various compounds that were intended to control the passage of fluid across the capillaries or the cell membranes. Following perfusion the albumin and EDTA space of each kidney and its water and cation content was measured. Total water content increased significantly unless 7–8% ($\text{w}\text{v}$) polyethylene glycol or dextran of mean MW 6000 was present in the perfusate. The EDTA space always increased, but this may have been due, at least in part, to penetration of the intracellular space by the marker. The albumin space was increased only in the presence of hydroxyethyl starch or sucrose. All the kidneys gained sodium and lost potassium, but this effect was least with the perfusate containing dextran of MW 6000 which also gave the lowest EDTA space, a normal total water content, and a normal vascular resistance. It is suggested that the addition of low molecular weight polymers to perfusates used for organ preservation will help to control edema, which may result in improved function after transplantation.     

Hepatic organic anion transport kinetics and bile flow during short-term total parenteral nutrition in the rabbit

Plasma disappearance of sulfobromophthalein (BSP) after an intravenous bolus (5 mg/kg) was determined in six lab chow-fed (LCF) rabbits and in six rabbits maintained on total parenteral nutrition (TPN) for 5 days. A common bile duct cannula enabled measurements of bile flow and biliary BSP excretion. Compartmental analysis of the biexponential plasma disappearance curve yielded three fractional transfer rates, plasma to liver (hepatic uptake), liver to plasma (reflux), and liver to bile (canalicular excretion). The transfer rates for hepatic uptake were 0.253 +/- 0.061/min for LCF and 0.147 +/- 0.040/min for TPN (P less than 0.01) and for the canalicular excretion of BSP were 0.038 +/- 0.019/min for LCF and 0.019 +/- 0.002/min for TPN (P less than 0.05). Model-computed rates for BSP excretion in bile over 60 min were lower with TPN (61%) than with LCF (80%); the measured excretory rates were 53% for TPN rabbits and 75% of injected dose for LCF animals. Basal biliary flow was reduced by 50% in the TPN group. With a two-compartmental model, assuming two pools and three transfer rates, we have demonstrated for the first time significant decreases in hepatic uptake and canalicular excretion of the organic anion BSP during TPN. A decrease in hepatic blood flow due to the enteral fast of TPN could have contributed in part to the decreased hepatic uptake. But, because the second exponent of the biexponential curve is independent of hepatic blood flow, the decrease in liver to bile transfer rate is a true approximation of a diminished canalicular excretory capacity during TPN. It is concluded that the movement of organic anions along the hepatic BSP/bilirubin transport system is impaired early during TPN.     

Mutations in novel organic cation transporter (OCTN2), an organic cation/carnitine transporter, with differential effects on the organic cation transport function and the carnitine transport function

Novel organic cation transporter (OCTN2) is an organic cation/carnitine transporter, and two missense mutations, L352R and P478L, in OCTN2 have been identified as the cause for primary carnitine deficiency. In the present study, we assessed the influence of these two mutations on the carnitine transport function and the organic cation transport function of OCTN2. The L352R mutation resulted in a complete loss of both transport functions. In contrast, the P478L mutation resulted in a complete loss of only the carnitine transport function but significantly stimulated the organic cation transport function. Studies with human OCTN2/rat OCTN2 chimeric transporters indicated that the carnitine transport site and the organic cation transport site were not identical. Because carnitine transport is Na(+)-dependent whereas organic cation transport is Na(+)-independent, we investigated the possibility that the P478L mutation affected Na(+) binding. The Na(+) activation kinetics were found to be similar for the P478L mutant and wild type OCTN2. We then mutated nine different tyrosine residues located in or near transmembrane domains and assessed the transport function of these mutants. One of these mutations, Y211F, was found to have differential influence on the two transport activities of OCTN2 as did the P478L mutation. However, the Na(+) activation kinetics were not affected. These findings are of clinical relevance to patients with primary carnitine deficiency because whereas each and every mutation in these patients is expected to result in the loss of the carnitine transport function, all of these mutations may not interfere with the organic cation transport function.     

Parathion and methyl parathion toxicity and metabolism in piperonyl butoxide and diethyl maleate pretreated mice

Pretreatment of male mice with piperonyl butoxide, 400 mg/kg 1 h before challenge with insecticides, resulted in a 40-fold antagonism of the acute i.p. toxicity of methyl parathion but potentiated the toxicity of parathion two-fold. Piperonyl butoxide had no effect on the toxicity of the oxygen analogs of these insecticides, methyl paraoxon and paraoxon. Diethyl maleate (1 ml/kg) depleted liver glutathione by 80% after one hour, potentiated the toxicity of both methyl parathion and methyl paraoxon, and partially counteracted the protective effect of piperonyl butoxide on methyl parathion toxicity. Piperonyl butoxide delayed the onset of brain cholinesterase inhibition by parathion. Studies of the metabolism of the insecticides by liver homogenates in vitro demonstrated that piperonyl butoxide inhibited both the oxidative formation of the oxygen analogs (activation) and oxidative cleavage to p-nitrophenol and dialkylphosphorothioic acid (detoxification). While parathion metabolism was mostly oxidative, methyl parathion metabolism appeared to be predominantly via glutathione-dependent enzymes. Studies of in vitro distribution of the insecticides demonstrated that piperonyl butoxide pretreatment resulted in elevated tissue concentrations of parathion and methyl parathion; however, the rate constant for elimination from plasma for both insecticides was unaffected by piperonyl butoxide. The overall rate of metabolism of methyl parathion in vivo was approximately twice that of parathion. These results suggest that during piperonyl butoxide inhibition of oxidative activation and cleavage, methyl parathion detoxification continues through uninhibited glutathione-dependent pathways of metabolism. The net result is a reduction in the acute toxicity of methyl parathion. Lack of an effective alternate pathway of detoxification may explain the delayed but greater toxicity of parathion in piperonyl butoxide pretreated mice.     

Effect of temperature and the synergist piperonyl butoxide on imidacloprid toxicity to the cat flea (Siphonaptera: Pulicidae)

The toxicity of imidacloprid to cat fleas on glass was investigated at 20, 26, 30, and 35 degrees C. Imidacloprid was most toxic to adult cat fleas at 35 degrees C and to larvae at 20 degrees C. Piperonyl butoxide (PBO), a synergist, increased the relative potency of imidacloprid (1:5 imidacloprid:PBO) 16-fold at 26 degrees C against adults, but had no effect at 35 degrees C. No synergism occurred in larvae at 20 degrees C, but addition of PBO (1:5 imidacloprid:PBO) doubled toxicity at 26 degrees C. PBO (1:5 imidacloprid:PBO) could possibly be used to synergize imidacloprid premise treatments (20-30 degrees C), but it is not likely to be effective in pet treatments because no synergism occurred in adult fleas at 35 degrees C (average fur temperature of tested cats and dogs).     

Regulation of organic anion transport in the liver.

In several liver diseases the biliary transport is disturbed, resulting in, for example, jaundice and cholestasis. Many of these symptoms can be attributed to altered regulation of hepatic transporters. Organic anion transport, mediated by the canalicular multispecific organic anion transporter (cmoat), has been extensively studied. The regulation of intracellular vesicular sorting of cmoat by protein kinase C and protein kinase A, and the regulation of cmoat-mediated transport in endotoxemic liver disease, have been examined. The discovery that the multidrug resistance protein (MRP), responsible for multidrug resistance in cancers, transports similar substrates as cmoat led to the cloning of a MRP homologue from rat liver, named mrp2. Mrp2 turned out to be identical to cmoat. At present there is evidence that at least two mrp''s are present in hepatocytes, the original mrp (mrp1) on the lateral membrane, and mrp2 (cmoat) on the canalicular membrane. The expression of mrp1 and mrp2 in hepatocytes appears to be cell-cycle-dependent and regulated in a reciprocal fashion. These findings show that biliary transport of organic anions and possibly other canalicular transport is influenced by the entry of hepatocytes into the cell cycle. The cloning of the gene for cmoat opens up new possibilities to study the regulation of hepatic organic anion transport.