The effects of intracage ventilation on microenvironmental conditions in filter-top cages.

Filter-top cages, while effective in reducing cross contamination by particulate material including microbes, can also cause accumulation of the waste gases carbon dioxide and ammonia as well as increased intracage relative humidity. A prototype system which provided each cage with 23 air changes per hour through a nozzle inserted in the filter lid was evaluated. The ventilated cageing system was effective in reducing intracage carbon dioxide, ammonia and relative humidity levels. Mean weekly carbon dioxide levels were 2900 ppm lower, ammonia levels 240 ppm lower and intracage relative humidity levels 8% lower in the ventilated cages than in unventilated controls.     

A forced-air individually ventilated caging system for rodents

The practice of using a protective filter apparatus when housing five mice per cage resulted in ammonia levels exceeding 25 ppm. A forced-air individually ventilated caging system was constructed using polyvinyl-chloride tubing fitted to a rodent rack. The ammonia level was decreased using this ventilation system.     

Evaluation of individually ventilated cage systems for laboratory rodents: occupational health aspects

New ventilated caging systems for laboratory animals were compared with conventional caging regarding allergen distribution, ergonomic suitability, cage environment and animal welfare. This paper presents occupational health evaluations. Mice were placed in individually ventilated cage (IVC) systems, a ventilated cabinet, and in cages on open shelves (conventional husbandry). The IVC systems were studied at negative and positive airflow. Aeroallergens were sampled on filters (n = 204, including controls) in undisturbed rooms and during cage changing. Concentrations of mouse urinary allergen (Mus m 1) in filter eluates were measured using sandwich ELISA. An ergonomic evaluation was performed with measurement of traction forces. Staff exposure during cage changing was high in all systems, range 116-4430 ng Mus m 1/m3. In undisturbed animal rooms, allergen levels were orders of magnitude higher when using conventional caging compared with ventilated systems; P < 0.001. At positive pressure both IVCs leaked allergen (median Mus m 1 concentration was < 0.08 ng/m3 at negative, but 6.5 ng/m3 (IVC1) and 0.8 ng/m3 (IVC2S) at positive pressure). The IVC systems had ergonomic disadvantages compared with the conventional husbandry and the ventilated cabinet, for instance with cages in unsuitable working heights. Ventilated husbandry solutions reduce levels of airborne allergen substantially at negative pressure, but are ergonomically less suitable. To prevent allergen exposure during cage changing, we propose that this procedure should be performed under ventilated conditions. Producers and users must cooperate in optimizing animal caging systems for both animals and staff.     

Biochemical stress indicators of greater wax moth exposure to organophosphorus insecticides

Although acetylcholinesterase (AChE) is the primary target of organophosphorus insecticides (OPs), increasing evidence regarding their secondary effects suggests that OPs disturb homeostasis of insects by generating free radical intermediates that trigger lipid peroxidation. We therefore investigated alterations in lipid peroxidation product, malondialdehyde (MDA) content, and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, in conjunction with AChE activity as biochemical stress indicators in greater wax moth, Galleria mellonella (L.) larvae for OPs methyl parathion (MP) and ethyl parathion (EP). The effects of MP and EP were first investigated by rearing the young larvae on an artificial diet containing 0.01, 0.1, 1, 10, and 100 ppm of each insecticide. Second, the mature larvae were injected with 0.05, 0.5, 5, 50, and 500 ng of insecticides for determining the changes in biochemical stress responses. The diet with lowest level of MP significantly decreased the activities of all measured enzymes, whereas it increased MDA content. However ALT and AST were significantly higher in the larvae reared with the diet with high levels of MP than in control larvae. All tested levels of MP resulted in a decrease in AChE activity. The lowest level of EP in diet (0.01 ppm) significantly increased ALT activity, whereas it reduced that of AChE. This insecticide at 0.1 ppm resulted in reduced AST activity, but 1 ppm in diet elevated AST activity and MDA content. EP at 0.1 ppm and higher levels in the diet reduced ALT activity. All dietary EP levels significantly decreased AChE activity. ALT, AST, and AChE were lower in larvae fed with the diet containing 100 ppm ethyl parathion compared with larvae on control diet. MP at 50 ng per larva increased ALT and AST activities from 35.42 +/- 0.74 and 26.34 +/- 0.83 to 203.57 +/- 1.09, and 122.90 +/- 1.21 U/g, respectively, when the mature larvae were injected. All injected doses of EP dramatically reduced both ALT and AST activities, but only the lowest and highest levels of this insecticide decreased AChE activity. The lowest level of this insecticide also significantly increased MDA content in larvae. High levels of both insecticides increased MDA content. We observed a significant higher increase in MDA content in the larvae reared with 10 ppm EP (102.16 +/- 1.57 nmol/g protein) than the control group (30.28 +/- 1.42 nmol/g protein). These results suggest that OPs caused the metabolic and synaptic dysfunctions in greater wax moth and alter its biochemical physiology in response to oxidative stress.     

Cage ammonia levels during murine reproduction.

Monogamous pairs of inbred BALB/c and outbred CD-1 mice housed in M2 cages with a floor area of 330 cm2, produced a mean cage ammonia level of 26 ppm and 154 ppm respectively over a four day period prior to weaning their litters. A gradient of ammonia exists from the nest to the food hopper. By housing CD-1 monogamous pairs in RM2 cages which have double the floor area of M2 cages (676 cm2), a lower mean level of ammonia was recorded at the same stage of reproduction and air sampling. The CD-1 mice in particular, were subjected to high levels of ammonia when compared with long term human health and safety occupational exposure limits of 25 ppm.     

The calm mouse: an animal model of stress reduction

Chronic stress is associated with negative health outcomes and is linked with neuroendocrine changes, deleterious effects on innate and adaptive immunity, and central nervous system neuropathology. Although stress management is commonly advocated clinically, there is insufficient mechanistic understanding of how decreasing stress affects disease pathogenesis. Therefore, we have developed a "calm mouse model" with caging enhancements designed to reduce murine stress. Male BALB/c mice were divided into four groups: control (Cntl), standard caging; calm (Calm), large caging to reduce animal density, a cardboard nest box for shelter, paper nesting material to promote innate nesting behavior, and a polycarbonate tube to mimic tunneling; control exercise (Cntl Ex), standard caging with a running wheel, known to reduce stress; and calm exercise (Calm Ex), calm caging with a running wheel. Calm, Cntl Ex and Calm Ex animals exhibited significantly less corticosterone production than Cntl animals. We also observed changes in spleen mass, and in vitro splenocyte studies demonstrated that Calm Ex animals had innate and adaptive immune responses that were more sensitive to acute handling stress than those in Cntl. Calm animals gained greater body mass than Cntl, although they had similar food intake, and we also observed changes in body composition, using magnetic resonance imaging. Together, our results suggest that the Calm mouse model represents a promising approach to studying the biological effects of stress reduction in the context of health and in conjunction with existing disease models.     

Expression of arabidopsis cytosolic ascorbate peroxidase gene in response to ozone or sulfur dioxide

The effects of ozone or sulfur dioxide on antioxidant enzymes were investigated in Arabidopsis thaliana. Plants were fumigated with 0.1–0.15 ppm ozone or sulfur dioxide up to about 1 week in an environment-controlled chamber. Both pollutants increased the activities of ascorbate peroxidase and guaiacol per-oxidase in leaves, but had little effect on the activities of superoxide dismutase, catalase, monodehydroascorbate reductase, dehydroascorbate reductase or glutathione reductase. Ozone was more effective than sulfur dioxide in increasing the activities of the peroxidases. Ascorbate peroxidase activity increased 1.8-fold without a lag period during fumigation with 0.1 ppm ozone, while guaiacol peroxidase activity increased 4.4-fold with a 1-day lag. Expression of the APX1 gene encoding cytosolic ascorbate peroxidase was further investigated. Its protein levels in leaves exposed to 0.1 ppm ozone for 4 or 8 days were 1.5-fold higher than in controls. Both ozone and sulfur dioxide elevated APX1 mRNA levels in leaves at 4 and 7 days, whereas at 1 day only ozone was effective. The induction of APX1 mRNA levels by ozone (3.4- to 4.1-fold) was more prominent than that by sulfur dioxide (1.6-to 2.6-fold). The APX1 mRNA level increased by day and decreased by night. Exposure of plants to 0.1 ppm ozone enhanced the APX1 mRNA level within 3 h, which showed a diurnal rhythm similar to that of the control. These results demonstrate that near-ambient concentrations of ozone as well as similar concentrations of sulfur dioxide can induce APX1 gene expression in A. thaliana.Environmental Biology Division     

Simultaneous determination of residual tetracyclines in foods by high-performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry

We established a method for precisely determining residual tetracycline antibiotics (TCs) in foods by atmospheric pressure chemical ionization liquid chromatography-tandem mass spectrometry (APCI LC–MS–MS) using selected reaction monitoring with an internal standard. By setting the nebulizer probe temperature to 475°C, we were able to use a mobile phase containing oxalic acid without clogging problems at the APCI interface, since oxalic acid decomposes to carbon dioxide and water at high temperature. DMCTC was very effective as an internal standard for determining TCs in various foods. TCs were cleaned up using a Bond Elut ENV cartridge and analysed by APCI LC–MS–MS. The recovery of TCs from various foods including animal tissues, honey, milk, eggs, and fish fortified at levels of 0.05, 0.10, and 0.50 ppm averaged 60.1–88.9%, with an RSD of 1.2–8.7%. The detection limits were 0.001 ppm for OTC and TC, 0.004 ppm for CTC, and 0.002 ppm for DC. The present method was also successfully used to determine TCs in swine kidney samples that were previously found by microbiological assay.     

A filtration method for measuring cellular uptake of [14C]methylamine and other highly permeant solutes

A filtration technique has been developed for study of the uptake of [14C]methylamine by Azotobacter vinelandii. This dual filter arrangement requires a precision microporous polycarbonate film which overlays a paper filter. Cellular uptake of radioactivity is terminated by vacuum filtration of the reaction mixture onto the polycarbonate filter without dilution or washing. Filtration was complete in 0.7 s with retention of less than 0.2% of the extracellular radioactivity. The dual filter method gave 20-fold higher levels for intracellular methylamine than filtration followed by washing. Without washing, nitrocellulose filters retained 18 times more extracellular [3H]sorbitol and 80 times more extracellular [14C]methylamine than polycarbonate filters. Use of an underlying paper filter did not significantly improve the performance of nitrocellulose filters. However, addition of a paper filter reduced extracellular methylamine and sorbitol retention on polycarbonate filters by 77 and 86%, respectively. This method is generally applicable to measurement of the uptake of highly permeant molecules by cells and subcellular organelles.     

Effect of NO2 on Airborne Venezuelan Equine Encephalomyelitis Virus

Studies were conducted to determine the effect of nitrogen dioxide (NO(2)) on aerosol survival and biological decay rate of Venezulean equine encephalomyelitis (VEE) virus and spores of Bacillus subtilis var. niger. The NO(2) concentrations used in the experiments were 0.5, 5, and 10 ppm at 24 C and 85% RH. The survival of airborne VEE virus disseminated as particles 1 to 5 mum in diameter was significantly influenced by the presence of 5 ppm of NO(2). At this concentration, the biological decay rate increased threefold and the aerosol recovery and aerosol survival of the VEE virus were significantly lower than at 0.5 ppm or in the absence of NO(2). Airborne spores of B. subtilis were not significantly affected by as much as 10 ppm of NO(2).     

Efficacy of ozone fumigation for laboratory animal sanitation]

Ozone resistance of spores of 2 strains of Bacillus isolated from laboratory animals was examined. Each of 0.02 ml of phosphate-buffered saline at pH 7.0 containing 10(6) Bacillus spores was dropped onto sterilized filter strips, wood chip bedding, pellets of diet, cloth pieces and stainless steel plates. After drying at room temperature, the test materials were exposed to ozone gas of different concentrations at 90% RH. Exposure to 200ppm ozone for 6 hours was sufficient to kill spores in filter strips, but a little higher concentration or a little longer period of ozone fumigation was necessary for sterilization of wood chips, cotton cloth pieces and steel plates. The present results indicated that 600ppm ozone fumigation for 6 hours might be effective for routine sterilization of cages, wood bedding, working clothes and other materials used in laboratory animal facilities. However, exposure to ozone gas of 500 or 1,000 ppm for 6 hours or 200 ppm for 24 hours could not kill spores in pellets of diet, suggesting that dietary protein inhibited the bactericidal activity of ozone.     

Microclimate in two types of rat cages

The microclimate in two types of rat cages (a Makrolon type IV with a solid floor and a stainless steel cage with a wire mesh floor (five rats per cage)) placed in the same macro-environment was compared. The temperature, relative humidity and ammonia concentration in the cages were measured twice a day for 8 days. The cages were cleaned every 4 days. The greatest difference between the cage types was in the ammonia build-up. In Makrolon cages the ammonia concentration never reached 5 ppm, whereas in steel cages it showed a constant increase and already exceeded the threshold limit for man (25 ppm for 8 h per day) on the third day after cleaning.     

Removal of a high load of ammonia by a marine bacterium,Vibrio alginolyticus in biofilter

A newly isolated heterotrophic marine bacterium,Vibrio alginolyticus, was used to remove a high load of ammonia gas under non-sterile condition. The cells were inoculated onto an inorganic packing material in a fixed-bed reactor (biofilter), and a high bad of ammonia, in the range of ammonia gas concentration of 170 ppm to 880 ppm, was introduced continuously. Sucrose solution and 3% NaCl was supplied intermittently to supplement the carbon source and water to the biofilter. The average percentage of gas removed exceeded 85% for 107-day operation. The maximum removal capacity and the complete removal capacity were 19 g-N kg⁻¹ dry packing material day⁻¹ and 16 g-N kg⁻¹ dry packing material day⁻¹, respectively, which were about three times greater than those obtained in nitrifying sludge inoculated onto the same packing material. On day 82, the enhanced pressure drop was restored to the normal one by NaOH treatment, and efficient removal characteristics were later observed. During this operation, the non-sterile condition had no significantly adverse effect on the removability of ammonia byV. alginolyticus.     

Ammonia as an attractive component of host odour for the yellow fever mosquito, Aedes aegypti

Behavioural responses of Aedes aegypti mosquitoes to ammonia were investigated in a modified Y-tube olfactometer. Ammonia was attractive in concentrations from 17 ppb to 17 ppm in air when presented together with lactic acid. Aqueous solutions of ammonia salts in concentrations comparable to those found in human sweat also increased the attractiveness of lactic acid. The role of lactic acid as an essential synergist for ammonia became further apparent by the fact that ammonia alone or in combination with carbon dioxide was not effective, even though the synergistic effect of carbon dioxide and lactic acid was corroborated. An extract from human skin residues, which attracts approximately 80% of the tested mosquitoes, contains both lactic acid and ammonia. The combination of these compounds, however, attracts no more than 45%, indicating that other components on human skin also play a role in host finding. Preparative liquid chromatography of the skin extract yielded three behaviourally active fractions which work together synergistically. Fraction III contains lactic acid as the effective principle; the compositions of the other two have not been clarified yet. The attractiveness of fraction I was augmented considerably when ammonia was added, whereas the effect of fraction II was not influenced by ammonia. These results suggests that ammonia is part of the effective principle of fraction II and contributes to the attractive effect of host odours.     

The impact of reduced frequency of cage changes on the health of mice housed in ventilated cages

Our purpose in this investigation was to determine if we could reduce cage changing frequency without adversely affecting the health of mice. We housed mice at three different cage changing frequencies: 7, 14, and 21 days, each at three different cage ventilation rates: 30, 60 and 100 air changes per hour (ACH), for a total of nine experimental conditions. For each condition, we evaluated the health of 12 breeding pairs and 12 breeding trios of C57BL/6J mice for 7 months. Health was assessed by breeding performance, weanling weight and growth, plasma corticosterone levels, immune function, and histological examination of selected organs. Over a period of 4 months, we monitored the cage microenvironment for ammonia and carbon dioxide concentrations, relative humidity, and temperature one day prior to changing the cage. The relative humidity, carbon dioxide concentrations, and temperature of the cages at all conditions were within acceptable levels. Ammonia concentrations remained below 25 ppm (parts per million) in most cages, but, even at higher concentrations, did not adversely affect the health of mice. Frequency of cage changing had only one significant effect; pup mortality with pair matings was greater at the cage changing frequency of 7 days compared with 14 or 21 days. In addition, pup mortality with pair matings was higher at 30 ACH compared with other ventilation rates. In conclusion, under the conditions of this study, cage changes once every 14 days and ventilation rates of 60 ACH provide optimum conditions for animal health and practical husbandry.     

棉铃虫蛹期在极端湿度下的失水动态

研究了极端相对湿度 (0%、9%、22.5%、80%、90%和100%) 对棉铃虫Helicoverpa armigera蛹期发育、存活和水分动态的影响。发育蛹在25℃下,相对湿度≤9%时, 不能羽化;湿度为22.5%时,羽化率不足20%; 高湿不影响它们的存活。在同样温度下,湿度≥9%时,滞育蛹在一个月内都极少死亡;在此期间,滞育终止率随湿度降低而升高。各湿度处理组发育蛹和滞育蛹从1日龄起的累计失水率都与其日龄呈线性相关。三个低湿处理组发育蛹中死亡个体在死亡前的平均累计失水率都在32%以上。滞育蛹经0%湿度处理一个月,平均仅失水22.4%;在湿度≥90%时的同期失水率不超过3.6%。在30℃下,发育蛹在4 h内测定的表皮渗透力最大,分别是9.0(♀)和10.7(♂) μg/(cm²·h·mm Hg); 滞育蛹的相应值出现在2 h内, 分别为 4.7(♀)和5.4(♂) μg/(cm²·h·mm Hg)。     

Interactions of ecdysteroid and juvenoid agonists in Plodia interpunctella (Hübner)

The influence of non-steroidal ecdysteroid agonists on Indianmeal moth larvae was assessed by rearing last instar larvae on diet treated with RH-5992 (tebufenozide) or RH-2485 (methoxyfenozide). Larvae were monitored for effects of the ecdysteroid agonists on weight, metamorphosis and mortality. Larvae treated with either of the ecdysteroid agonists at a concentration of 5 ppm or higher gained less weight and had greater mortality than did larvae reared on control diet. For example, the weights of control larvae increased approximately 400% by day 2, compared with only a 50% increase in weight when the larvae were treated with 25 ppm of RH-2485 or RH-5992. Similarly, mortality in control larvae was less than 10%, but was as much as 90–100% in larvae reared on diet treated with one of the ecdysteroid agonists. We also examined the effects of simultaneous treatment with a juvenile hormone (JH) mimic, either methoprene or fenoxycarb. The JH mimics prevented adult emergence, and the larvae continued to feed throughout the month-long observation period. However, larvae treated with a juvenile hormone mimic gained weight despite the presence of an ecdysteroid agonist in the diet. On diets treated with 0.1 ppm of RH-2485 or RH-5992, JH-treated larvae gained even more weight than did untreated controls. Interestingly, although the addition of a JH mimic to ecdysteroid-treated diet resulted in increased weight, it did not lead to reduced mortality. In fact, combinations of a JH mimic with 10 ppm RH 2485 or RH 5992 resulted in nearly 100% mortality compared with 40–70% mortality without the JH compounds. These results indicate that JH mimics overcome the inhibitory effects of ecdysteroid agonists on weight gain; however, they also resulted in increased mortality compared with moderate doses of ecdysteroid agonists alone. One specific action of these compounds at the cellular level was noted in that RH 5992 mimicked ecdysteroids by increasing uptake of ¹⁴C-GlcNAc in a Plodia interpunctella cell line, while fenoxycarb was inhibitory. Arch. Insect Biochem. Physiol. 38:91–99, 1998. Published 1998 Wiley-Liss, Inc.     

Nitrogen dioxide air pollution near ambient levels is an atherogenic risk primarily in obese subjects: a brief communication

Ambient exposure to nitrogen dioxide, a critical air pollutant in developed countries, is positively associated with cardiovascular mortality and morbidity. Although its cardiovascular effects are predominantly shown in patients with high risk of atherogenesis, no studies have elucidated whether daily exposure to nitrogen dioxide air pollution enhances atherogenic metabolisms, primarily in obese subjects who are susceptible to atherogenesis and subsequent cardiovascular diseases. We used male Otsuka Long-Evans Tokushima Fatty (OLETF) rats as obese subjects and Long-Evans Tokushima (LETO) rats as nonobese controls. The animals were continuously exposed to nitrogen dioxide at a concentration of 0, 0.16, 0.8, or 4.0 ppm from 8 weeks of age through 32 weeks. At 40 weeks of age, levels of body weight, triglyceride, and total cholesterol were significantly greater in the OLETF rats than in the LETO rats. A ratio of high-density lipoprotein (HDL) to total cholesterol was significantly smaller in the former than in the latter. In the LETO rats, nitrogen dioxide exposure significantly decreased only the levels of HDL as compared with clean air exposure. In the OLETF rats, however, nitrogen dioxide exposure at a concentration of 0.16 ppm significantly elevated triglyceride concentration and decreased the ratio of HDL to total cholesterol as well as the levels of HDL. Nitrogen dioxide air pollution near ambient levels is an atherogenic risk primarily in obese subjects.     

DNA damage repair in quiescent murine mammary carcinoma cells in culture

Murine mammary carcinoma cells (line 67) were grown in unfed cultures for up to 9 days. In cultures (day 2-3) in which cells were proliferatively active and in day 3-5 (transition) cells, a large fraction of nuclear DNA was retained on polycarbonate filters when assayed by the alkaline filter elution technique. In contrast, the fraction of DNA retained on filters was significantly reduced for nonproliferating (Q, quiescent) cells from unfed 7-9 day cultures. The increase in endogenous DNA breaks followed both the decrease in proliferative state and clonogenicity in these cells. When day 7 Q cells were refed these endogenous DNA breaks were removed with a half-time of about 2.5 h. When the cells were exposed to X-irradiation and the integrity of their nuclear DNA measured by the alkaline filter elution assay, as much as a 2-fold greater frequency of radiation-induced DNA breaks was produced in Q versus P cells. DNA breaks were also removed from irradiated Q cells at a rate which was 0.23 that observed in P cells. We suggest that the depressed capacity for DNA damage removal in Q cells is responsible for their greater radiosensitivity, and the impaired DNA damage repair is probably due to a reduced level of energy sources in these unfed Q cell cultures.     

Biotreatment of ammonia- and butanal-containing waste gases

The biological removal of ammonia and butanal in contaminated air was investigated by using, respectively, a laboratory-scale filter and a scrubber-filter combination. It was shown that ammonia can be removed with an elimination efficiency of 83% at a volumetric load of 100 m³·m^–2·h^–1 with 4–16 ppm of ammonia. During the experiment percolates were analysed for nitrate, nitrite, ammonium and pH. It was found that the nitrification in the biofilter could deteriorate due to an inhibition of Nitrobacter species, when the free ammonia concentration was rising in the percolate. It should be easy to control such inhibition through periodic analysis of the liquid phase by using a filter-scrubber combination. Such a combination was studied for butanol removal. Butanal was removed with an elimination efficiency of 80% by a scrubber-filter combination at a volumetric load of 100 m³·m^–2·h^–1 and a high butanal input concentration. Mixing the filter material with CaCO₃ and pH control of the liquid in the scrubber resulted in an increase of the elimination efficiency. These results, combined with previous results on the biofiltration of butanal and butyric acid, allow us to discuss the influence of odour compounds on the removal efficiency of such systems and methods for control. The results were used to construct a full-size system, which is described.