Ole Ladefoged

Lung inflammation and genotoxicity following pulmonary exposure to nanoparticles in ApoE -/- mice

BackgroundThe toxic and inflammatory potential of 5 different types of nanoparticles were studied in a sensitive model for pulmonary effects in apolipoprotein E knockout mice (ApoE-/-). We studied the effects instillation or inhalation Printex 90 of carbon black (CB) and compared CB instillation in ApoE-/- and C57 mice. Three and 24 h after pulmonary exposure, inflammation was assessed by mRNA levels of cytokines in lung tissue, cell composition, genotoxicity, protein and lactate dehydrogenase activity in broncho-alveolar lavage (BAL) fluid.ResultsFirstly, we found that intratracheal instillation of CB caused far more pulmonary toxicity in ApoE-/- mice than in C57 mice. Secondly, we showed that instillation of CB was more toxic than inhalation of a presumed similar dose with respect to inflammation in the lungs of ApoE-/- mice. Thirdly, we compared effects of instillation in ApoE-/- mice of three carbonaceous particles; CB, fullerenes C60 (C60) and single walled carbon nanotubes (SWCNT) as well as gold particles and quantum dots (QDs). Characterization of the instillation media revealed that all particles were delivered as agglomerates and aggregates. Significant increases in Il-6, Mip-2 and Mcp-1 mRNA were detected in lung tissue, 3 h and 24 h following instillation of SWCNT, CB and QDs. DNA damage in BAL cells, the fraction of neutrophils in BAL cells and protein in BAL fluid increased statistically significantly. Gold and C60 particles caused much weaker inflammatory responses.ConclusionOur data suggest that ApoE-/- model is sensitive for evaluating particle induced inflammation. Overall QDs had greatest effects followed by CB and SWCNT with C60 and gold being least inflammatory and DNA-damaging. However the gold was used at a much lower mass dose than the other particles. The strong effects of QDs were likely due to Cd release. The surface area of the instilled dose correlated well the inflammatory response for low toxicity particles.

The acute effects of mono(2-ethylhexyl)phthalate (MEHP) on testes of prepubertal Wistar rats

A single oral dose of 400 mg/kg body weight of mono(2-ethylhexyl)phthalate (MEHP), the testis toxic metabolite of di(2-ethylhexyl)phthalate, was given to 28-day-old male Wistar rats and the testis toxic effects were investigated 3,6, and 12 h after exposure. Detachment and sloughing of germ cells were observed, and in the Sertoli cells the cytoplasmatic intermediate filament vimentin collapsed. In the immunohistochemical investigation the androgen receptor distribution was unchanged between the control group and treated groups. The expression of the testosterone-repressed-prostatic-message-2 gene in rat testis increased after 3 h, but returned to control levels after 6 and 12 h. Caspase-3 activity increased 3 and 12 h after MEHP exposure. This increase could not be correlated to an increase in DNA fragmentation or increase in apoptotic numbers of germ cells. In conclusion, the effect of MEHP in testis is apparently not involving the androgen receptor. Vimentin localisation in the Sertoli cells, and increased levels of caspase-3 activity appear to be sensitive and early markers of MEHP testis toxicity.

Di(2-ethylhexyl) adipate (DEHA) induced developmental toxicity but not antiandrogenic effects in pre- and postnatally exposed Wistar rats

Di(2-ethylhexyl) adipate (DEHA) has replaced the phthalates in thin plasticized polyvinyl chloride films used for food packaging, mainly because some phthalates induce testis toxicity and antiandrogenic effects. A dose-range finding study followed by a dose-response/effect study in Wistar rats investigated whether pre- and postnatal DEHA doses of 0, 800, or 1200mg/kg/day body weight and doses of 0, 200, 400, or 800mg/kg/day (main study) elicited developmental toxicity including antiandrogenic effects. In the main study, DEHA induced a prolonged gestation period (800mg/kg/day) and a dose-related increase in postnatal death (400 and 800mg/kg/day). DEHA also induced a permanent decrease in offspring body weight (800mg/kg/day). No antiandrogenic endpoints were affected. We conclude that DEHA induced developmental toxicity and the NOAEL is 200mg/kg. DEHA did not induce antiandrogenic effects similar to those of di(2-ethylhexyl) phthalate even though the chemical structures have similarities and the two chemicals have a common metabolite.

Developmental toxicity of toluene in male rats: effects on semen quality, testis morphology, and apoptotic neurodegeneration.

Abstract. In one study, pregnant Wistar rats were exposed to 1200xa0ppm toluene by inhalation 6 h a day from gestational day (GD) 7 to postnatal day (PND) 18. Sperm analysis was performed in the adult male offspring at PND 110 by using computer-assisted sperm analysis. Toluene did not affect the semen quality of exposed rats. In another study, pregnant rats were exposed to 1800xa0ppm from GD 7 to GD 20, and the male offspring were killed at PND 11, 21 or 90. Paired testes weight, histopathology and immunoexpression of vimentin in Sertoli cells were used as markers of testis toxicity. In the brain, the number of apoptotic cells in the hippocampus and cerebellum were counted after visualisation by means of the TUNEL assay. Mean body weight in pups of exposed dams was lower than in pups from control litters. This decrease was still statistically significant at PND 11, but at PND 21 and 90 the body weight of toluene-exposed males tended to approach that of the controls. Absolute and relative testes weights were reduced in all three age groups, although not to a statistically significant degree. Histopathological examinations of the testis and immuno-expression of vimentin did not reveal any differences between toluene-exposed animals and control animals. In the hippocampus, almost no apoptosis was observed in any age group, and there were no differences in apoptotic neurodegeneration between male rats exposed to 1800xa0ppm and control animals at PND 11, 21 or 90. Generally, a marked increase in number of apoptotic cells was observed in cerebellar granule cells at PND 21 compared with the other age groups. Toluene induced a statistically significant increase in the number of apoptotic cells in the cerebellar granule layer at PND 21. The mean was increased from 37 in the control group to 71 in the toluene-exposed group. Thus, the granular cell layer in cerebellum is a highly relevant tissue with which to study toluene-induced apoptosis, because of the continuous migration of neurons and high frequency of neuronal apoptosis during the weaning period. In summary, it is concluded, that neither pre- and postnatal exposure to 1200xa0ppm toluene nor prenatal exposure to 1800xa0ppm induced significant effects on the reproductive parameters investigated. However, prenatal exposure to 1800xa0ppm toluene did increase neuronal apoptosis in the cerebellum of weaned male rats, possibly by delaying postnatal migration of granule cells to their final destination, or by toluene-induced retardation of generalised fetal growth.

Probabilistic cumulative risk assessment of anti-androgenic pesticides in food

In this paper, we present a cumulative risk assessment of three anti-androgenic pesticides (vinclozolin, procymidone and prochloraz) using the relative potency factor (RPF) approach and an integrated probabilistic risk assessment (IPRA) model. RPFs for each substance were estimated for three reproductive endpoints (ano-genital distance, and weights of the seminal vesicles and the musculus levator ani/bulbocavernosus) in male rat foetuses exposed in utero. The cumulative dietary intake was estimated based on consumption data and residue data from the Netherlands. The IPRA model combines variability in both exposure and sensitivity between individuals into a distribution of individual margins of exposures (IMoEs) and IMoEs of 1 or less indicate a possible concern. The assessment did not result in IMoEs < or = 1. The endpoint weight of seminal vesicles resulted in the lowest IMoEs (0.1th percentile: 198) and the fraction of individuals with IMoEs<1000 was 1.43%. For the two other endpoints, the fractions were slightly lower. Thus, cumulative dietary exposure of Dutch women to vinclozolin, procymidone and prochloraz is not likely to be of concern for the reproductive development of their male foetuses. However, other anti-androgenic substances and exposure routes should also be included in the cumulative assessment to make it more comprehensive.

Report on a conference on organic solvents and the nervous system

At two scientific conferences in 1985, one in Copenhagen sponsored by the Nordic Council of Ministers and the World Health Organization (WHO), the other in Raleigh, NC, it was concluded that chronic toxic encephalopathy may develop following long-term occupational exposure to organic solvents (1,2). The terms organic affective syndrome, mild and severe chronic toxic encephalopathy were suggested for this condition describing increasing severity. In May 1990, a conference on organic solvents and the nervous system was held in Copenhagen sponsored by the Commission of the European Communities and the Danish Ministry of the Environment (3). Scientists and representatives from the governments, industries, and labour organisations from the EEC and US participated.

Dearomatized white spirit inhalation exposure causes long-lasting neurophysiological changes in rats

Exposure for 6 h per day, 5 days per week, during a period of 6 months to the organic solvent dearomatized white spirit (0, 400, and 800 ppm) was studied in rats that were 3 months old when the repeated exposure was initiated. After an exposure-free period of 2-6 months duration, neurophysiological, neurobehavioral, and macroscopic pathologic examinations were performed. The study revealed exposure-related changes in sensory evoked potentials and a decrease in motor activity during dark (no light) periods but no white spirit-induced changes in learning and memory functions. The measurements of the flash evoked potential (FEP), somatosensory evoked potential (SEP), and auditory brain stem response (ABR) all demonstrated dose-dependent increases of the amplitudes of the early latency peaks of the sensory evoked potentials (EPs). Furthermore, an increase of the dose showed that the measurements of FEP and SEP revealed changes in the later-latency peaks, which reflect the more associative aspects of sensory processing. The results demonstrated that 6 months of exposure to dearomatized white spirit induced long-lasting and possible irreversible effects in the nervous system of the rat.

Toxicity study of di(2-ethylhexyl)phthalate (DEHP) in combination with acetone in rats

In two separate studies with exposure duration 9 weeks or 4 weeks, male Wistar rats were dosed with di(2-ethylhexyl)phthalate (DEHP) by gavage and exposed to drinking water with or without acetone (0.5% wt/v in the 9-week study, 1.0% wt/v in the 4-week study). In the 9-week study the doses of DEHP were 0, 125, 250, 500 or 1000 mg/kg b.wt. In the 4-week study the doses of DEHP were increased to 1000, 5000 and 10,000 mg/kg b.wt. In the 9-week study, the relative liver weight was increased in the rats exposed to 500 and 1000 mg/kg b.wt. No interaction of DEHP and acetone was observed in any of the measured parameters. In the 4-week study DEHP, at the highest dose level, resulted in severe general toxicity. The group exposed to DEHP in combination with acetone was more affected. Male fertility was decreased. Body weight was decreased, and the relative weight of the liver, kidney, heart, brain and adrenals increased. The relative weight of the testes decreased in the 5000 and 10,000 mg/kg b.wt. groups. The weight of seminal vesicles and epididymals decreased at 10,000 mg/kg b.wt. In animals exposed to 5000 and 10,000 mg DEHP/kg b.wt. a severe atrophy of the seminiferous tubules and a slight diffuse Leydigs cell hyperplasia was observed. The cellular debris and conglomerates of desquamated cells found in the lumen of the seminiferous tubules were immunostained positive for vimentin. This indicates that Sertoli cell cytoplasm is included in the conglomerates an interesting finding not previously described. No specific interaction of DEHP and acetone was observed in any of the measured parameters.

Changes in markers of oxidative status in brain, liver and kidney of young and aged rats following exposure to aromatic white spirit

Levels of glutathione and activity of glutamine synthetase were assayed in organs of rats following inhalation of a heterogeneous solvent mixture containing both aliphatic and aromatic hydrocarbons. This mixture was administered for 3 weeks (6 h daily) at two levels in the inhaled air (400 and 800 ppm) to young adult (5-month-old) and aged (14-month-old) rats. Depression of levels of glutamine synthetase in the P2 fraction of kidney was observed, which was more severe in aged than young adult rats. Glutamine synthetase is a cytosolic enzyme especially susceptible to oxidative damage. A parallel depression of this enzyme was also seen in the corresponding hepatic fractions. However, levels of glutamine synthetase in the hippocampus were elevated by this exposure. Glutathione levels were depressed in P2 fractions of livers of exposed rats, and also in the corresponding renal fraction. Glutathione concentration was unchanged in cerebral fractions. Overall results were interpreted to imply that pro-oxidant events were elevated in kidney and liver following prolonged inhalation of the solvent mixture. The changes found in brain tissue did not reveal evidence of oxidative stress but, however, suggested that glial activation was taking place.

Three weeks' and six months' exposure to aromatic white spirit affect synaptosomal neurochemistry in rats

The effects of 3 weeks or 6 months inhalation exposure of rats to aromatic white spirit 6 h/day, 5 days/week at 0, 400, or 800 ppm were studied. Synaptosomal neurochemistry was investigated as index of the in situ conditions in the presynaptic nerve terminal. In both studies, the relative and absolute yield of synaptosomal protein were significantly reduced in the two exposed groups. Both studies demonstrated increased synaptosomal noradrenaline (NA), dopamine (DA), and 5-hydroxytryptamine (5-HT) concentrations, high- affinity 5-HT uptake rate and uptake capacity. It is hypothesized that a reduced density and total number of synapses in situ may be functionally compensated by increased NA, DA, and 5-HT neurotransmitter release, or by increased activity of corresponding neurons. The increased synaptosomal 5-HT uptake rates and uptake capacities may explain the previously demonstrated increased global and regional neurotransmitter concentrations and the present finding of increased synaptosomal 5-HT concentrations. These changes are interpreted as an indication of toxic effect on the CNS function and are considered supportive of recent findings of electrophysiological changes and affected motor activity following 6 months exposure to dearomatized white spirit followed by an exposure-free period.