Airway exposure to multi-walled carbon nanotubes disrupts the female reproductive cycle without affecting pregnancy outcomes in mice.

BACKGROUND: The use of multiwalled carbon nanotubes (MWCNT) is increasing due to a growing use in a variety of products across several industries. Thus, occupational exposure is also of increasing concern, particularly since airway exposure to MWCNTs can induce sustained pulmonary acute phase response and inflammation in experimental animals, which may affect female reproduction. This proof-of-principle study therefore aimed to investigate if lung exposure by intratracheal instillation of the MWCNT NM-400 would affect the estrous cycle and reproductive function in female mice. RESULTS: Estrous cycle regularity was investigated by comparing vaginal smears before and after exposure to 67 µg of NM-400, whereas reproductive function was analyzed by measuring time to delivery of litters after instillation of 2, 18 or 67 µg of NM-400. Compared to normal estrous cycling determined prior to exposure, exposure to MWCNT significantly prolonged the estrous cycle during which exposure took place, but significantly shortened the estrous cycle immediately after the exposed cycle. No consistent effects were seen on time to delivery of litter or other gestational or litter parameters, such as litter size, sex ratio, implantations and implantation loss. CONCLUSION: Lung exposure to MWCNT interfered with estrous cycling. Effects caused by MWCNTs depended on the time of exposure: the estrous stage was particularly sensitive to exposure, as animals exposed during this stage showed a higher incidence of irregular cycling after exposure. Our data indicates that MWCNT exposure may interfere with events leading to ovulation.

Ambient and at-the-ear occupational noise exposure and serum lipid levels.

OBJECTIVES: Occupational and residential noise exposure has been related to increased risk of cardiovascular disease. Alteration of serum lipid levels has been proposed as a possible causal pathway. The objective of this study was to investigate the relation between ambient and at-the-ear occupational noise exposure and serum levels of total cholesterol, low-density lipoprotein-cholesterol, high-density lipoprotein-cholesterol, and triglycerides when accounting for well-established predictors of lipid levels. METHODS: This cross-sectional study included 424 industrial workers and 84 financial workers to obtain contrast in noise exposure levels. They provided a serum sample and wore portable dosimeters that every 5-s recorded ambient noise exposure levels during a 24-h period. We extracted measurements obtained during work and calculated the full-shift mean ambient noise level. For 331 workers who kept a diary on the use of a hearing protection device (HPD), we subtracted 10 dB from every noise recording obtained during HPD use and estimated the mean full-shift noise exposure level at the ear. RESULTS: Mean ambient noise level was 79.9 dB (A) [range 55.0-98.9] and the mean estimated level at the ear 77.8 dB (A) [range 55.0-94.2]. Ambient and at-the-ear noise levels were strongly associated with increasing levels of triglycerides, cholesterol-HDL ratio, and decreasing levels of HDL-cholesterol, but only in unadjusted analyses that did not account for HPD use and other risk factors. CONCLUSION: No associations between ambient or at-the-ear occupational noise exposure and serum lipid levels were observed. This indicates that a causal pathway between occupational and residential noise exposure and cardiovascular disease does not include alteration of lipid levels.

Evaluation of microbial qPCR workflows using engineered Saccharomyces cerevisiae.

AIMS: We describe the development and interlaboratory study of modified Saccharomyces cerevisiae as a candidate material to evaluate a full detection workflow including DNA extraction and quantitative polymerase chain reaction (qPCR). METHODS AND RESULTS: S. cerevisiae NE095 was prepared by stable insertion of DNA sequence External RNA Control Consortium-00095 into S. cerevisiae BY4739 to convey selectivity. For the interlaboratory study, a binomial regression model was used to select three cell concentrations, high (4 × 10(7) cells ml(-1)), intermediate (4 × 10(5) cells ml(-1)) and low (4 × 10(3) cells ml(-1)), and the number of samples per concentration. Seven participants, including potential end users, had combined rates of positive qPCR detection (quantification cycle <37) of 100%, 40%, and 0% for high, intermediate, and low concentrations, respectively. CONCLUSIONS: The NE095 strain was successfully detected by all participants, with the high concentration indicating a potential target concentration for a reference material. SIGNIFICANCE AND IMPACT OF THE STUDY: The engineered yeast has potential to support measurement assurance for the analytical process of qPCR, encompassing the method, equipment, and operator, to increase confidence in results and better inform decision-making in areas of applied microbiology. This material can also support process assessment for other DNA-based detection technologies.

Prenatal and adult stress interplay--behavioral implications.

The origin of adult behavior and the possible pathogenesis of psychiatric disorders remain elusive, but extensive research indicates that interaction of genes and environment play a crucial role for adult phenotype. Differences in susceptibility may arise by earlier experiences and genomic variables, either alone or in combination. The acoustic startle response (ASR) has been shown to be altered in patients with several psychiatric diseases, a change that could result from a persistent sensitization caused by chronic arousal secondary to a traumatic incident. The current work hypothesized that a single aversive procedure would induce long-term hyperactivity in the HPA-axis of rats that had become vulnerable by prenatal stress, and thereby change reactivity in the ASR. Prenatal stress was achieved by maternal gestational exposure to Chronic Mild Stress (CMS). At age 3 months, the offspring were blood sampled by a stressful procedure, and subsequently tested in the acoustic startle paradigm. Prenatal CMS strongly reduced prepulse inhibition (PPI) whereas postnatal blood sampling under restraint generally increased PPI. Our data demonstrate interplay between pre- and postnatal stressful events, but also that this interaction is complex and could influence the interplay between PPI and basal startle. Our results suggest that circumstances dating back to early development may have implications for adult life behavior, and based on this we propose a new theory of a threshold in the induction of a stress response in the ASR test, which influences whether the PPI or basal startle response will be affected.

Hearing loss and cochlear damage in experimental pneumococcal meningitis, with special reference to the role of neutrophil granulocytes.

Hearing loss is a well-known sequelae from meningitis, affecting up to 25% of survivors. However, the principal components of the infectious and inflammatory reaction responsible for the sensorineural hearing loss remain to be identified. The present study aimed to investigate the impact of an augmented neutrophil response on the development of hearing loss and cochlear damage in a model of experimental pneumococcal meningitis in rats. Hearing loss and cochlear damage were assessed by distortion product oto-acoustic emissions (DPOAE), auditory brainstem response (ABR) and histopathology in rats treated with ceftriaxone 28 h after infection. Rats were treated with Granulocyte Colony Stimulating Factor (G-CSF) initiated prior to infection, 28 h after infection or with ceftriaxone only. Rats were followed for 7 days, and assessment of hearing was performed before infection and 24 h and day 8 after infection. Pretreatment with G-CSF increased hearing loss 24 h after infection and on day 8 compared to untreated rats (Mann-Whitney, P = 0.012 and P = 0.013 respectively). The increased sensorineural hearing loss at day 8 was associated with significantly decreased spiral ganglion cell counts (P = 0.0006), increased damage to the organ of Corti (P = 0.007), increased areas of inflammatory infiltrates (P = 0.02) and increased white blood cell (WBC) counts in cerebrospinal fluid on day 8 after infection (P = 0.0084). Initiation of G-CSF 28 h after infection did not significantly affect hearing loss or cochlear pathology compared to controls. In conclusion, the inflammatory host reaction contributes significantly to the development of hearing loss in experimental meningitis.

Toxic encephalopathy and noise-induced hearing loss.

In several laboratory animal studies, it has been documented that the hearing, vision, and brain can be injured due to exposure to organic solvents. This finding formed the background for a pilot study (n=16) aimed at identifying new ways of qualifying diagnostics, treatment, and rehabilitation of patients suffering from brain injury due to exposure to organic solvents, also referred to as toxic encephalopathy. Diagnosing toxic encephalopathy is complicated because the symptoms of this type of diffuse brain injury are non-specific. So, it was initially hypothesised that some of the difficulties involved in diagnosing toxic encephalopathy could be minimized by extending the diagnostic procedure. Apart from clinical interviewing and neuropsychological testing, the diagnosis should include the examination of hearing and vision. This will help in achieving new measures that could improve in diagnosing toxic encephalopathy with more certainty. On the basis of ranking, only one patient in the pilot study was considered to have a normal neuropsychological test profile, which was defined as a test profile without any marked deviations when compared with a normal population. A total of 10 patients were considered to have "discrete problems." These patients had a test profile showing either a few strikingly negative results or an array of results slightly below the expected level when compared with a normal population. A total of four patients were considered to suffer from "moderate problems" and one patient from "severe problems." The patients with "moderate problems" and "severe problems" showed consistent negative results and an unambiguous negative test profile. However, the overall results of all neuropsychological examinations performed revealed a dispersed picture. Quite remarkably, all the 13 patients who had their hearing examined showed a loss of hearing, 7 patients complained about tinnitus, and all patients had a history of exposure to both noise and organic solvents, which had not been observed at the initial examination, but seemed to have serious implications for their prognosis and future life.

Behavioural effects in rats after prenatal exposure to dearomatized white spirit.

The purpose of the study was to investigate the potential developmental neurotoxicity of the widely used organic solvent, white spirit. Rats (Mol:WIST) were exposed to 0 or 800 ppm dearomatized white spirit for 6 hr per day on gestation days 7-20. Developmental and neurobehavioural effects in the offspring were investigated using a test battery including assessment of physical development, reflex ontogeny, motor function, motor activity and, learning and memory. No significant effects were recorded on motor function and the activity in Open Field. In the initial learning period (age 1 month), the performance in a Morris water maze was similar in exposed and control animals. When testing for memory at the age of 2 months, the exposed male offspring used more time to locate the hidden platform. After platform relocation, impaired cognitive function was revealed in the exposed females. At the age of 5 months, learning and memory deficits were observed in exposed offspring. The differences were not related to poorer swimming capabilities, because swim speeds were similar to control values. The results show that prenatal exposure to 800 ppm white spirit caused long-lasting learning and memory deficits in rats.

Four weeks' inhalation exposure of Long Evans rats to 4-tert-butyltoluene: effect on evoked potentials, behaviour, and brain neurochemistry.

Long-lasting central nervous system (CNS) neurotoxicity of 4-tert-butyltoluene (TBT) has been investigated using electrophysiology, behaviour, and neurochemistry in Long Evans rats exposed by inhalation to 0, 20, or 40 p.p.m. TBT 6 hr/day, 7 days/week for 4 weeks. Flash evoked potentials and somatosensory evoked potentials were not affected by TBT. In Auditory Brain Stem Response there was no shift in hearing threshold, but the amplitude of the first wave was increased in both exposed groups at high stimulus levels. Three to four months after the end of exposure, behavioural studies in Morris water maze and eight-arm maze failed to demonstrate any TBT induced effects. Exposure was followed by a 5 months exposure-free period prior to gross regional and subcellular (synaptosomal) neurochemical investigations of the brain. TBT reduced the NA concentration in whole brain minus cerebellum. Synaptosomal choline acetyltransferase activity increased and acetylcholinesterase activity was unchanged suggesting increased synaptosomal ability for acetylcholine synthesis. The relative and total yield of synaptosomal protein was reduced suggesting reduced density and total number of synapses in situ, respectively. We hypothesise that a reduced yield of synaptosomal protein reflects a more general effect of organic solvent exposure on the software of the brain. The synaptosomal concentration per mg synaptosomal protein and the total amount of 5-hydroxytryptamine were not affected whereas the total amount of synaptosomal noradrenaline decreased. The concentration and the total amount of synaptosomal dopamine decreased. The noradrenergic and dopaminergic parts of CNS may be more vulnerable to TBT than the serotonergic, and these long-lasting effects may cause or reflect TBT-compromised CNS function.

Developmental neurotoxicity after toluene inhalation exposure in rats.

Rats were exposed to 1200 ppm or 0 ppm toluene (CAS 108-88-3) for 6 h per day from day 7 of pregnancy until day 18 postnatally. Developmental and neurobehavioral effects in the offspring were investigated using a test battery including assessment of functions similar to those in the proposed OECD TG for Developmental Neurotoxicity Study, i.e., physical development, reflex ontogeny, motor function, motor activity, sensory function, and learning and memory. The exposure did not cause maternal toxicity or decreased viability of the offspring. Lower birth weight, delayed ontogeny of reflexes, and increased motor activity in the open field was registered in the exposed offspring. Impaired cognitive function was revealed in the exposed female offspring at the age of 3.5 months, i.e., they used more time to locate the hidden platform in the Morris water maze after platform relocation. The difference was not related to poorer swimming capabilities, because swim speeds were similar to control values. The results show that exposure to 1200 ppm toluene during brain development caused long-lasting developmental neurotoxicity in rats.

Effects of prenatal exposure to toluene on postnatal development and behavior in rats.

Development and neurobehavioral effects of prenatal exposure to toluene (CAS 108-88-3) were studied after exposing pregnant rats (Mol:WIST) to 1800 ppm of the solvent for 6 h daily on days 7-20 of gestation. Body weights of exposed offspring were lower until day 10 after parturition. Neurobehavioral evaluation of the pups revealed no effects on motor function (rotarod), activity level (open field), acoustic startle, and prepulse inhibition. Measurements of hearing function using auditory brain stem response revealed small effects in male-exposed offspring. Performance in a Morris water maze during initial learning gave some indications of impaired cognitive functions, which was confirmed during further testing, especially in reversal and new learning. Effects on cognitive functions seemed most marked in female offspring.

Toxicity of the styrene metabolite, phenylglyoxylic acid, in rats after three months' oral dosing.

Male Wistar rats were dosed with 0, 1250, 3750 or 5000 mg/l of phenylglyoxylic acid (PGA) (CAS no. 611-73-4) in the drinking water ad libitum for 3 months. During the entire treatment period, there were no gross signs of toxicity related to PGA. No changes in neurobehavior were found after using a functional observational battery or radial arm maze. An increased relative kidney weight was seen in the highest dose-group (Controls: 0.504 +/- 0.031 g/100 g b.wt.; 5000 mg PGA/l: 0.579 +/- 0.033 g/100 g b.wt.; p<0.01). No other organ weights were affected. Histopathology revealed no change in kidney structure. No changes in clinical biochemistry. In the highest dose-group three animals out of ten showed reduction in peripheral nerve myelin sheath thickness. No such changes were seen in the control group. The study revealed no changes in auditory brain stem response but minor changes in electroretinography. The noradrenaline (NA) concentration decreased in pons and thalamus whereas it increased in medulla oblongata and whole brain. The dopamine (DA) concentration increased in cerebellum, hippocampus, pons, and whole brain. The most marked DA increase was seen in hippocampus (Controls: 0.56 +/- 0.10 nmol/g tissue; 5000 mg/l: 1.04 +/- 0.11 nmol/g tissue; p<0.001). The 5-hydroxytryptamine (5-HT) concentration decreased in cerebellum, cerebral cortex, hippocampus, and medulla oblongata, whereas it increased in thalamus. The yield of synaptosomal protein, synaptosomal NA, DA, and 5-HT concentrations, and DA uptake rate were not affected. When dosed males were mated with naive females, there were no differences between groups in the pregnancy rate, number of corpora luteae, implantations, live or dead fetuses, resorptions, preimplantation loss, or postimplantation loss. It is concluded that a part of the effects on kidney, peripheral nerves, and vision, which have previously been reported after exposure to styrene, might be induced by the styrene metabolite, PGA. If PGA has ototoxic effects in rats, the dosing in the present study is not sufficient to induce the necessary ototoxic concentration in blood. Alternatively, the ototoxicity of styrene, like toluene, may be caused the parent compound itself and not by a metabolite like PGA.

Dietary structured triacylglycerols containing docosahexaenoic acid given from birth affect visual and auditory performance and tissue fatty acid profiles of rats.

To examine whether it is possible to enhance the level of 22:6(n-3) in the central nervous system, newborn rats were fed dietary supplements containing oils with either specific or random triacylglycerol structure, but similar concentrations of polyunsaturated fatty acids. In the specific structured oil, 22:6(n-3) was located in the sn-2 position, whereas it was equally distributed among the three positions in the triacylglycerol molecule in the randomized oil. A reference group was fed rat milk before weaning and nonpurified diet after weaning. After 12 wk, the levels of 22:6(n-3) in brain and liver phospholipids were higher in the groups fed the experimental diets than in the reference group. The specific structured oil resulted in the highest level of 22:6(n-3) in the brain, whereas the level of 22:6(n-3) was highest in the liver of the group fed randomized oil, indicating differences in metabolism of fatty acids resulting from their position in the dietary triacylglycerol molecule. The higher levels of 22:6(n-3) were accompanied by significantly lower levels of the long-chain (n-6) polyunsaturated fatty acids compared with the reference group. The fatty acid profiles, including the level of 22:6(n-3), in the retina phospholipids were not affected by the three different diets apart from a lower level of 20:4(n-6) in rats fed the experimental diets, indicating a strong tendency to maintain a high level of 22:6(n-3) in the retina. The changes in the fatty acid profiles did not result in differences in learning ability, but caused changes in visual function, evidenced by higher latency of the b-wave and lower oscillatory potential, and in auditory brainstem response, evidenced by generally greater amplitude of wave Ia in the group fed specific structured oil.

Long-lasting neurobehavioral effects of prenatal exposure to xylene in rats.

The persistence of neurobehavioral effects in female rats (Mol:WIST) exposed to 500 ppm technical xylene (dimethylbenzene, CAS-no 1330-20-7) for 6 hours per day on days 7-20 of prenatal development was studied. The dose level was selected so as not to induce maternal toxicity or decreased viability of offspring. Investigations of learning and memory abilities were performed using a Morris water maze. This task requires rats to spatially navigate, using distal extramaze cues to locate a small platform under the surface of the water in a large pool. At the age of 16 weeks, the exposed offspring showed impairments when the platform was relocated in the pool. Impaired performances after platform relocation were also observed in exposed offspring at 28 and 55 weeks of age, although the difference was not statistically significant at 55 weeks. These data could indicate that the effect was partly reversible, although over a long time period. However, another explanation could be that the animals became more practised at solving the problem (finding the platform) as continued testing occurred and therefore were able to compensate for the neurotoxic effect of the prenatal xylene exposure. Further studies are planned to investigate whether neurobehavioral effects resulting from prenatal xylene exposure can interact with neurophysiological aging processes.

Four weeks' inhalation exposure of rats to p-cymene affects regional and synaptosomal neurochemistry.

Long-lasting effects of inhalation exposure to p-cymene (p-isopropyl-toluene; CAS No. 99-87-6) on regional and subcellular brain neurochemistry were studied. Male Long-Evans rats were exposed to 0, 50, or 250 p.p.m. p-cymene 6 hr/day, 5 days/week for four weeks followed by an exposure-free period of 8 weeks. Synaptosomes were isolated from whole brain minus cerebellum and used as an ex situ model for in situ conditions at the level of the presynaptic nerve terminal. There was no persistent effect on wet weight (regional) or regional noradrenaline (NA), dopamine (DA), or 5-hydroxytryptamine (5-HT) concentrations owing to exposure. Yield of synaptosomal protein was statistically significantly reduced in an exposure concentration-related manner (Control: 16.6 +/- 3.1; 50 p.p.m.: 9.2 +/- 2.1; 250 p.p.m.: 8.6 +/- 1.7 mg protein/g tissue, mean +/- I.S.D.). Synaptosomal NA and DA concentrations and acethycholinesterase, butyrylcholinesterase, and lactate dehydrogenase activities were statistically significantly increased when expressed relative to synaptosomal protein. It is hypothesized that a reduced density and number of synapses in situ are functionally compensated for by increased NA and DA release from noradrenergic and dopaminergic presynaptic nerve terminals. The applicability of the synaptosome as an ex situ neurochemical research model for the presynaptic CNS nerve terminal in situ for the study of solvent neurotoxicity in rats was further supported.

An approach to risk assessment.

A strategy for delineating risk factors from use of neurotoxic chemicals was applied to the Danish working environment. An analysis using this strategy disclosed the need for internationally adopted criteria for neurotoxicity, and consequently a working group was established by the Nordic Council of Ministers with the task to propose criteria for neurotoxicity. Functional effects on the nervous system, such as reduction in memory and learning ability, decrease in attention, and alteration of behavior due to toxic chemicals in the environment is now being acknowledged as an important public health problem. This change in concern from obvious effects of high dose exposure to the more subtle effects of environmental exposure to neurotoxicants was incorporated in the criteria. The new criteria for evaluating neurotoxicity data has subsequently been used on 79 common industrial chemicals. The results indicate that numerous persons are exposed in the working as well as in the general environment to several chemicals, for which almost no data on the effect on subtle neurophysiological functions are available. Development of an approach to risk assessment dealing with this problem is a major challenge in the nineties. Different approaches to risk assessment are discussed, the quality of the databases available for hazard assessment are evaluated, and the needs for further research are identified.

The effect of ageing and in vitro exposure to xylene and KCl on [Ca2+]i in synaptosomes from rats exposed prenatally to xylene.

Female rats (Mol: WIST) were exposed prenatally to 500 p.p.m. of technical xylene on days 7-20. At the age of fourteen months the rats were sacrificed and the synaptosomal fraction prepared for in vitro studies. The cytosolic calcium concentration was measured using the FURA-2 technique. The cytosolic calcium was increased in synaptosomes from old rats compared to those from rats at the age of three months, but no effect of prenatal exposure was seen. When synaptosomes were incubated with xylene, potassium or both, the cytosolic calcium concentration was changed identically in all groups of rats. When synaptosomes were incubated simultaneously to xylene and potassium a dramatical leakage of FURA-2 was observed. The mechanisms behind the membrane leakage are discussed.

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.

Effects of prenatal exposure to xylene on postnatal development and behavior in rats.

The effects of prenatal exposure to the organic solvent xylene (dimethylbenzene, CAS-no 1330-20-7) on postnatal development and behavior in rats were studied. Pregnant rats (Mol:WIST) were exposed to 500 ppm technical xylene 6 h per day on gestation days 7-20. The dose level was selected so as not to induce maternal toxicity or decrease the viability of offspring. In the exposed offspring, a delay in the ontogeny of the air righting reflex, a lower absolute brain weight, and impaired performance in behavioral tests for neuromotor abilities (Rotarod) and for learning and memory (Morris water maze) were found. Generally, the effects were most marked in the female offspring.

Two weeks inhalation exposure to 4-tert-butyltoluene causes persistent changes in visually evoked potentials in rats.

The effects of repeated exposure to 20 p.p.m. 4-tert-butyltoluene (CAS No. [98-51-1]) 6 hr/day for 14 days on the function of the intact nervous system were examined by measurements of flash evoked potentials in Wistar rats. The exposure to 4-tert-butyltoluene induced changes in the amplitudes of the flash evoked potentials. The changes were significantly different from controls on day 2, 19 and 26 after cessation of the exposure, but not on day 5 and 12. No significant difference in body weight gain between groups was found during the experiment. These results indicate that repeated exposure to 20 p.p.m. 4-tert-butyltoluene causes persistent changes in the function of the central nervous system measured as changes in the flash evoked potential. A reevaluation of the present TLV value of 10 p.p.m. for 4-tert-butyltoluene is suggested.