Oncogenicity studies of piperonyl butoxide in rats and mice

1. The oncogenicity of Piperonyl butoxide (PBO) has been studied in the mouse and rat. CD-1 mice were administered PBO in the diet at target doses of 0, 30, 100 and 300 mg/kg/day for 79 weeks and Sprague-Dawley rats 0, 30, 100 and 500 mg/kg/day for 104/105 weeks. 2. At termination of the study in the mouse there was evidence of increased liver weights and an increased incidence of eosinophilic adenomas at 100 and 300 mg/kg/day in males and 300 mg/kg/day in females. 3. In rats there was increased liver weights at 100 and 500 mg/kg/day associated with hepatocyte hypertrophy in both male and female rats. There was no increased incidence of neoplasia at any site. Hypertrophy and hyperplasia of thyroid follicles was observed at 500 mg/kg/day in both sexes. 4. The observations reflect the expected changes related to the induction of the mixed function oxygenase group of enzymes. In the mouse the increased incidence of eosinophilic adenomas is not considered relevant for human risk evaluation.     

Residue levels of pyrethrins and piperonyl butoxide in soil and runoff water

Simultaneous analysis of pyrethrins (Py-I and Py-II) and piperonyl butoxide (PBO) in soil and runoff water samples following field application of a new pyrethrum formulation containing pyrethrins (Py's) and PBO is described. Residues of total Py's and PBO were extracted from soil samples using hexane-acetone (9:1). A solid phase extraction (SPE) column containing C18-octadecyl bonded silica was used to separate Py's and PBO residues from runoff water. Residues in soil and water were quantitated by high performance liquid chromatography (HPLC) equipped with C18-column and a UV detector. Concentration of Py-II in soil was 100 times higher than that of Py-I 1 h following treatment and 9.6 times higher than Py-I in runoff surface water 11 days following treatment. Results indicated that Py's are non-persistent in soil (even though lipophillic) and water when applied at the recommended rate of 6 lbs (5.31 g A.I.) per acre. There was a consistent decrease in total Py's residues as time after spraying increased. Py's residues in soil decreased from 0.91 to 0.11 ppm 4 days following treatment and one month after treatment only 0.002 ppm were detected. The highest concentration of Py's in runoff water was 36.09 ng/liter following the first rainfall (11 days following treatment). PBO initial residues detected in soil samples were low (0.84 microgram/g soil) while no residues of PBO were detected in runoff water.     

Mechanistic study on liver tumor promoting effects of piperonyl butoxide in rats

Piperonyl butoxide, alpha-[2-(2-butoxyethoxy)ethoxy]-4,5-methylenedioxy-2-propyltol uene, is a widely used pesticide-synergist. Recently, results were reported indicating that piperonyl butoxide is a hepatocarcinogen in rat. Since the underlying mechanism was not elucidated, we examined the effects on rat liver cells in detail. For this purpose male F344 rats were administered piperonyl butoxide mixed in the diet at concentrations of 0 (negative control), 0.05, 0.2 or 2% for 2 days, 1, 2, and 4 weeks. As a positive control, phenobarbital was administered to rats for up to 4 weeks as a 0.1% solution in the drinking water. Increased liver weight, centrilobular hepatocellular hypertrophy due to increased smooth endoplasmic reticulum, decreased numbers and areas of connexin 32-positive spots per hepatocyte, and increased cell proliferation were observed in rats treated with 0.2 and 2% piperonyl butoxide. Similar results were obtained for 0.1% phenobarbital treated rats. Hepatocellular necrosis suggestive of hepatotoxicity was also observed in the 2% piperonyl butoxide group. These results indicate that the promoting mechanism of piperonyl butoxide in hepatocarcinogenesis is similar to that of phenobarbital, involving an ability to induce CYP isoenzymes and inhibit gap junctional intercellular communication. In addition, increased cell proliferation following hepatocellular necrosis may also play a role at high doses.     

Lack of effect of piperonyl butoxide on unscheduled DNA synthesis in precision-cut human liver slices

In this study the effect of piperonyl butoxide (PBO) on unscheduled DNA synthesis in precision-cut human liver slices has been examined. Liver slices prepared from tissue samples from five human donors were cultured in medium containing [3H]thymidine and 0-2.5 mM PBO using a dynamic organ culture system. After 24 h the liver slices were processed for autoradiographic examination of UDS. As positive controls, liver slices were also cultured with three known genotoxic agents, namely 2-acetylaminofluorene (2-AAF), aflatoxin B1 (AFB1) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). UDS was quantified as the net grain count in centrilobular hepatocytes and as the percentage of centrilobular hepatocyte nuclei with > 5 and > 10 net grains. Compared to control liver slice cultures PBO had no effect on UDS. In contrast, treatment with 0.02 and 0.05 mM 2-AAF, 0.002 and 0.02 mM AFB1 and 0.005 and 0.05 mM PhIP produced significant increases in net grain counts of centrilobular hepatocytes. The greatest induction of UDS was observed in liver slices treated with 0.05 mM PhIP. Treatment with 2-AAF, AFB1 and PhIP also produced increases in the number of centrilobular hepatocyte nuclei with > 5 and > 10 net grains. At the concentrations examined neither PBO, 2-AAF nor PhIP had any significant effect on replicative DNA synthesis in 24 h cultured human liver slices. In cultured liver slices treated with 0.02, but not 0.002, mM AFB1 a significant reduction in the rate of replicative DNA synthesis was observed. These results demonstrate that PBO does not induce UDS in cultured human liver slices. However, all three positive control compounds produced marked significant increases in UDS, thus confirming the functional viability of the human liver slice preparations used in this study. In conclusion, these results provide further evidence that PBO is a non-genotoxic agent which does not damage DNA in human liver.     

Dicationic furans inhibit development of Cryptosporidium parvum in HSD/ICR suckling Swiss mice

The efficacy of dicationic diarylfurans was evaluated against Cryptosporidium parvum by a suckling murine model. Candidate drugs were solubilized or suspended in deionized water and administered orally at a constant dose rate on days 0-5 (treatment day 0) to suckling ICR Swiss mice experimentally inoculated with oocysts of C. parvum. Efficacy was based on numbers of oocysts recovered from the intestinal tracts of mice subjected to necropsy examination on day 6. Numerous candidate furans significantly reduced the numbers of oocysts recovered from treated mice compared with control mice. Compounds 1, 2, 4, and 9 demonstrated superior efficacies (10% of controls or better) against C. parvum. Compounds 3, 5, 6, 7, 8, 11, 17, 18, and 19 also significantly reduced the numbers of oocysts recovered from treated mice but demonstrated efficacies ranging from 17 to 65% of controls. Compound 4 was particularly efficacious against C. parvum at a dosage as low as 8.5 mg/kg of body weight. Compound 4 is identified as a lead compound for additional studies in other animal models.     

Effects of diet on the lipid composition of Echinostoma caproni (Trematoda) in ICR mice

High-performance thin-layer chromatography (HPTLC) was used to determine neutral lipids and phospholipids in the intestinal trematode Echinostoma caproni from experimentally infected ICR mice fed a high-fat diet (hen's egg yolk) as compared with worms from mice fed a standard laboratory diet. Worms were removed from the hosts at 2, 3, and 4 weeks postinfection (p.i.). Analysis by TLC-densitometry showed significantly greater amounts of triacylglycerols and free sterols at 2, 3, and 4 weeks p.i. in worms from mice on the high-fat diet as compared with worms from mice on the standard laboratory diet. Significantly greater amounts of phosphatidylcholine and phosphatidylethanolamine were found in worms from mice on the high-fat diet as compared with worms from those on the standard diet at 2 weeks p.i. but not at 3 and 4 weeks p.i. The results of this study suggest that the host diet influences the lipid content of E. caproni adults.     

Developmental toxicity of 4-substituted amphetamines in mice

Until recently, therapy for most Nontuberculous Mycobacterial (NTM) disease, especially disease caused by Mycobacterium avium-complex (MAC), has been difficult and frustrating. The introduction of the newer macrolides (clarithromycin and azithromycin) has significantly added to the efficacy of regimens for both disseminated and pulmonary MAC disease. Clinical activity of these agents is lost when a MAC isolate develops in-vitro resistance that is facilitated by use of the macrolides as single agents. These valuable drugs should, therefore, never be used as single agents for the treatment of disseminated or pulmonary MAC disease. The newer macrolides also show promise for the treatment of other NTM infections such as those caused by M. abscessus, M. fortuitum, M. chelonae, M. xenopi, M. marinum, M. haemophilum, and M. genavense. Rifabutin, a derivative of rifamycin S, is an effective agent for prophylaxis against disseminated MAC and may have utility for treatment of pulmonary and disseminated MAC disease. Interactions between clarithromycin and rifamycin may alter efficacy of the macrolide and enhance toxicity of rifabutin. Although therapy of many NTM infections remains difficult with somewhat unpredictable results, the introduction of newer drugs, particularly the macrolides, has appreciably improved a previously dismal outlook for successful therapy.     

Subacute (4-wk) oral toxicity of a combination of four nephrotoxins in rats: comparison with the toxicity of the individual compounds

In a 4-wk study, 10-wk-old Wistar rats were fed the nephrotoxins hexachloro-1,3-butadiene (HCBD), mercuric chloride, d-limonene and lysinoalanine either alone or in combination. These nephrotoxins damage epithelial cells of the proximal tubules, but by different mechanisms. Each chemical was given alone at a Minimum-Nephrotoxic-Effect Level (MNEL), and at a No-Nephrotoxic-Effect Level (NNEL). The combination was given at the MNEL, the NNEL and one-quarter of the NNEL of the individual chemicals. The individual nephrotoxins caused slight growth depression in males at the MNEL, but not at the NNEL, whereas the combination depressed growth slightly at the NNEL and severely at the MNEL. In females at the MNEL, only HCBD retarded growth; in contrast to the effect in males this was not aggravated by combined treatment. Nephrotoxicity was more severe in males fed the combination than in males given the nephrotoxins alone. The former showed decreased renal concentrating ability and moderate histopathological changes in the kidneys at the MNEL, and a dose-dependent increase in kidney weight and number of epithelial cells in the urine at the NNEL and the MNEL. The males treated with a single agent showed slightly increased kidney weights, and/or slight histopathological changes in the kidneys at the MNEL, and (with d-limonene only) epithelial cells in the urine at the NNEL and MNEL. In females, renal changes induced by the combination were not more severe than those observed with individual compounds. No adverse changes attributable to treatment were observed in rats fed the combination at one-quarter of the NNEL. In the present study, combined exposure to four nephrotoxins at their individual NNEL did not constitute an obviously increased hazard, indicating absence of synergistic interaction, whereas at the MNEL clearly enhanced (renal) toxicity occurred in males, although not in females.     

Coenzyme Q10 and adriamycin toxicity in mice

Pretreatment for four days with coenzyme Q10 (CoQ10) significantly lowered the acute toxicity in female C3H/HeNCrlBR mice given moderately lethal (15.0 and 20.0 mg/kg) i.p. doses of adriamycin as well as in male ICR/Hla mice given 12.5 mg/kg i.p. adriamycin. In both strains of mice, CoQ10 pretreatment did not protect the mice at higher i.p. adriamycin dose levels. When adriamycin was administered by the clinically-used i.v. route, CoQ10 pretreatment did not reduce acute toxicity at moderately lethal doses in either strain. At higher i.v. adriamycin dose levels, CoQ10 pretreatment significantly enhanced acute toxicity. CoQ10 pretreatment did not alter the antitumor effectiveness of adriamycin (i.p. or i.v.) against the Dunn osteosarcoma.     

Acute toxicity and pharmacokinetics of dibekacin mice

The LD50 values of dibekacin to mice were determined following three different methods of administration, namely, intravenous constant infusion, intravenous bolus injection, and intramuscular injection. The serum levels of dibekacin were pharmacokinetically analyzed. The differences in LD50 values between the methods of administration were discussed from the viewpoints of pharmacokinetics. 1) The LD50 value following the intravenous constant infusion was higher than that following the intravenous bolus injection and approached the level of that following the intramuscular injection, when the infusion rate of the drug was decreased by increasing the infusion period. 2) The biological half-life of dibekacin in mice was 24--45 min. 3) The volume of distribution increased as its dose increased, and a linear correlation was noted between log Vd and log (dose). 4) The difference among the maximum serum concentrations calculated with dibekacin following the administration of LD50 was small, which coincided with the results of the experiment that the serum concentrations of dibekacin at the death following the administration of LD100 were almost the same regardless of the method of administration.     

Reduction of acetaminophen toxicity by sodium sulfate in mice

Twenty-one strains of Venezuelan encephalitis (VE) virus isolated from three habitats in Trinidad, W.I. during 1960 to 1972, were subtype III (Mucambo) VE virus by plaque-reduction neutralization tests. Like prototype Mucambo virus, each strain killed 8- to 15-week-old mice inoculated intraperitoneally. If the subtype I strain of VE virus that caused a major outbreak in Trinidad during 1943 to 1944 persisted on the island into the 1960s and early 1970s, it did not become the dominant VE virus in these three enzootic foci.     

Enhanced renal toxicity by inorganic mercury in metallothionein-null mice

To elucidate a protective role of metallothionein (MT) in the manifestation of inorganic mercury toxicity, we studied the susceptibility of MT-null mice to the renal toxicity of mercuric chloride. Because the MT-null (J) mice are a genetic background of 129/Sv strain, the 129/Sv mice were used as wild-type controls. Nine-week-old male MT-null (J) and 129/Sv mice were given subcutaneous injections of mercuric chloride at doses of 10 to 40 micromol/kg. The basal MT level in the kidney of MT-null (J) mice was undetectable (<0.2 microg/g of tissue) and approximately 2.5 microg/g of tissue in 129/Sv mice. The sensitivity to the renal toxicity of mercuric chloride was markedly enhanced in the MT-null (J) mice compared with the 129/Sv mice. The renal mercury level was similar for the MT-null (J) and 129/Sv mice at 4 hr after the injection of mercuric chloride (20 micromol/kg) but became significantly lower in MT-null (J) mice than in 129/Sv mice at 24 and 72 hr. Based on the present results, we conclude that MT is an important protective factor against the renal toxicity caused by inorganic mercury and that it may play a major role in the retention of mercury in the kidney.     

Developmental toxicity assessment of arsenic acid in mice and rabbits

This paper describes the intervention of glutathione-dependent enzymes, in particular the glutathione S-transferases (GSTs), in both the detoxication of electrophilic decomposition products resulting from the attack of oxygen radicals on lipids and DNA; and the prevention of oxygen toxicity generated by redox cycling catecholamine derivatives. The continuing growth of our knowledge of the glutathione S-transferase polygene family is described in terms of the increase in members of known gene families, the discovery of new ones and our increasing knowledge of their activities towards endogenous substrates.     

Susceptibility of heterozygous MnSOD gene-knockout mice to oxygen toxicity

OBJECTIVE: To study type IV collagen of skin and serum in patients with ALS. BACKGROUND: Collagen abnormalities of skin have been reported in ALS patients. However, little is known concerning type IV collagen in ALS. METHODS: We studied type IV collagen immunoreactivity of skin and measured serum levels of the 7S fragment of the N-terminal domain of type IV collagen (7S collagen) in patients with ALS and control subjects. RESULTS: The basement membrane as well as blood vessels of skin in ALS patients was weakly positive for type IV collagen as compared with those of diseased control subjects. This weak immunostaining became more pronounced as ALS progressed. The optical density for type IV collagen immunoreactivity in ALS patients was significantly lower (p < 0.001) than in diseased control subjects and was significantly decreased with duration of illness (r = -0.85, p < 0.01). Serum 7S collagen levels in patients with ALS were significantly decreased (p < 0.01) as compared with those in diseased and healthy control subjects and were negatively and significantly associated with duration of illness (r = -0.81, p < 0.001). There was an appreciable positive correlation between concentrations of serum 7S collagen and the density for type IV collagen immunoreactivity in ALS patients (r = 0.81, p < 0.02). CONCLUSIONS: These data suggest that a metabolic alteration of type IV collagen may take place in the skin of ALS patients and that the decreased levels of serum 7S collagen may reflect a decreased type IV collagen immunoreactivity of skin in patients with ALS.     

Comparative toxicity of azinphos-methyl to house mice, laboratory mice, deer mice, and gray-tailed voles

A laboratory toxicity study on house mice and laboratory mice (Mus musculus), gray-tailed voles (Microtus canicaudus), and deer mice (Peromyscus maniculatus) was conducted as part of a comprehensive laboratory and field study to field validate laboratory-based risk assessment of pesticides. The single dose oral LD50 for the organophosphorus insecticide azinphos-methyl (Guthion) was 10, 11, 32, and 48 mg/kg body weight in wild house mice, laboratory mice, gray-tailed voles, and deer mice, respectively. Ten-day dietary LC50s were 277 ppm for laboratory mice, 297 ppm for gray-tailed voles, and 1,180 ppm for deer mice. All treated animals lost more weight, consumed less food, and had depressed brain cholinesterase (ChE) activity compared to controls. Five-day LC50s were significantly higher than 10-day LC50s for laboratory mice and deer mice. For all three species, animals that died during dietary LC50 tests had mean ChE activity of 50-55% while survivors had 56-70% of controls. The conclusions were that: (1) Laboratory mice were not representative of deer mice or gray-tailed voles with respect to sensitivity to azinphos-methyl, but provided a conservative estimate for risk assessment; (2) 10-day dietary LC50 tests indicate substantially greater estimates of toxicity of azinphos-methyl to rodents than do 5-day tests; and (3) brain ChE depression of 45-50% was lethal in these species.     

Analgesic response in offspring of crosses between heroin delta (Swiss Webster) and mu (ICR) responding mice

To indirectly test the hypothesis whether serotonin (5-HT) might have a role in the increase in pulmonary vascular resistance, we evaluated the haemodynamic and gas exchange response of intravenous ketanserin (K), a 5-HT receptor inhibitor, in eight severe but stable patients with chronic obstructive pulmonary disease with secondary pulmonary hypertension (mean pulmonary artery pressure (Ppa) 30.3 +/- 7.3 mmHg). Measurements were done at baseline, after oxygen breathing (2 L.min-1), K bolus (6-15 mg) and finally during oxygen breathing (2 L.min-1) added to K infusion (3-6 mg.h-1). K bolus induced a significant reduction of mean Ppa (p < 0.05), mean systemic arterial pressure (p < 0.01) and total systemic resistance (p < 0.01). Cardiac index (+7%), oxygen delivery (+7%) and pulmonary vascular resistance (magnitude of the reduction: -12%) did not change significantly. When oxygen was added to K infusion, the cardiac index significantly dropped when compared to K bolus (p < 0.05), but oxygen delivery remained stable because of the resulting increase in arterial oxygen concentration; against baseline, the mean Ppa showed the same magnitude of reduction as with oxygen breathing or K bolus alone (p < 0.05). Ventilation and gas exchange were not significantly influenced by K bolus. When we individually analysed the changes of pulmonary vascular resistances by plotting the driving pressure through the pulmonary circulation against the cardiac output, we observed that an active vasodilating effect on the pulmonary circulation occurred with K in only one patient, while in three other patients there was rather a recruitment effect of the pulmonary vessels due to the systemic effects of the drug. In conclusion, this study of a small number of patients with severe chronic obstructive pulmonary disease associated with pulmonary hypertension shows that the parenterally given serotonin antagonist ketanserin predominantly affects the systemic circulation. Our results do not support the hypothesis that in stable chronic obstructive pulmonary disease patients with pulmonary hypertension, serotonin might have a role in the increase of pulmonary vascular tone.