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PubMed2001

Working with male rodents may increase risk of allergy to laboratory animals.

BACKGROUND: Our aim was to study the risk of laboratory animal allergy (LAA) among research staff working in laboratories separate from the animal confinement area. The roles of atopy and exposure intensity in LAA were studied with special regard to exposure to male rodents, who excrete higher levels of urinary allergens than female rodents. METHODS: Eighty rodent-exposed subjects gave blood samples for the analysis of total IgE, Phadiatop, and specific IgE against rat (RUA) and mouse urinary allergens (MUA), and answered questionnaires. Air samples were collected for RUA and MUA aeroallergen measurement in both laboratories and animal confinement facilities. RESULTS: Twenty percent of the subjects had IgE >0.35 kU/l to RUA and/or MUA, and 32% had experienced animal work-related symptoms, although 90% of aeroallergen samples from the research department laboratories were below the detection limit (<0.26 ng RUA per m(3) and <0.8 ng MUA per m(3)). Atopy (positive Phadiatop), total IgE >100 kU/l, other allergies (especially to other animals), or more than 4 years of exposure significantly increased laboratory animal sensitization and symptoms. Working with mainly male rodents gave odds ratios (95% CI) of 3.8 (0.97-15) for sensitization and 4.4 (1.4-14) for symptoms. Subjects with both exposure to mainly male rodents and atopy or elevated total IgE had a 10-fold higher frequency of sensitization than exposed subjects with neither risk factor. CONCLUSION: A majority of subjects with a combination of exposure to mainly male rodents and atopy or elevated total IgE developed sensitization to and symptoms from laboratory animals. Current low exposure seems to maintain the presence of specific IgE. Further measures must be undertaken to provide a safe workplace for laboratory animal workers.

Adult↗
PubMed1991

Polyamine catabolism in rodent and human cells in culture.

N1-Acetylspermidine (N1AcSpd) accumulates in late exponential phase, or after certain stresses such as heat shock, in both human tumour (A549) and rodent (HTC, CHO) cells, grown in medium containing an inhibitor of the FAD-dependent polyamine oxidase (PAO). Inhibition of PAO has little effect on cell growth or on the cellular content of the major polyamines, putrescine, spermidine or spermine, found in proliferating cells in culture, but decreases cellular putrescine content in heat shocked cells. Putrescine and spermidine are generated when N1AcSpd or N1-acetylspermine (N1AcSpm) respectively is added to either human or rodent cells depleted of the former amines by alpha-difluoromethylornithine. N1AcSpm is formed in polyamine-depleted human A549 cells when N1AcSpd is added to cultures treated with the PAO inhibitor. This reaction does not occur in either rodent line, suggesting that N1AcSpd can be converted directly into N1AcSpm in human, but not rodent, cells under specific conditions. The data presented demonstrate that a variety of human and rodent cells express PAO activity and catabolize polyamines by a mechanism which includes PAO. PAO activity is of little consequence to proliferating A549, HTC or CHO cells in culture, but does produce new putrescine in both late-exponential-phase and heat-shocked cells. These findings suggest that polyamine catabolism is part of a general response of both rodent and human cells to a variety of environmental and physiological stresses.

Animals↗
PubMed1975

South American mammal zoogeography: evidence from convergent evolution in desert rodents.

Current theories regarding colonization of South America by mammals are divided between those supported by fossil evidence, which suggest the original mammal fauna of the isolated continent was augmented by early immigrants (primates, caviomorph rodents, and later, procyonids) with a final large influx of northern mammals occurring with the formation of the Panama land bridge, and an opposing view which states that the purported "recent invaders" are too taxonomically and ecologically differentiated to have colonized since the land bridge arose. The second theory suggests that most extant mammals entered before the Plio-Pleistocene land connection. An analysis of degree of physiological adaptation, natural history, distribution patterns, and a multivariate assessment of convergent evolution of Monte Desert rodents indicate that South American cricetine rodents are not highly specialized for desert life. Their degree of adaptation could be accounted for, in large part, by adaptations for arid or semiarid Andean habitats. No Monte Desert rodent has developed the specialized desert traits that have evolved in most desert rodent faunas of the world, although extinct marsupials similar to living bipedal desert rodents were present in the Monte as recently as late Pliocene. Evidence suggests that Monte caviomorphs have been associated with the desert for a longer period than cricetines, and that the latter represent a fairly recent invasion of the Monte Desert. The data thus support the first hypothesis of South American mammal colonization.

Adaptation, Physiological↗
PubMed1985

Evidence for higher rates of nucleotide substitution in rodents than in man.

When the coding regions of 11 genes from rodents (mouse or rat) and man are compared with those from another mammalian species (usually bovine), it is found that rodents evolve significantly faster than man. The ratio of the number of nucleotide substitutions in the rodent lineage to that in the human lineage since their divergence is 2.0 for synonymous substitutions and 1.3 for nonsynonymous substitutions. Rodents also evolve faster in the 5' and 3' untranslated regions of five different mRNAs; the ratios are 2.6 and 3.1, respectively. The numbers of nucleotide substitutions between members of the beta-globin gene family that were duplicated before the man-mouse split are also higher in mouse than in man. The difference is, again, greater for synonymous substitutions than for nonsynonymous substitutions. This tendency is more consistent with the neutralist view of molecular evolution than with the selectionist view. A simple explanation for the higher rates in rodents is that rodents have shorter generation times and, thus, higher mutation rates. The implication of our findings for the study of molecular phylogeny is discussed.

Animals↗
PubMed2004

Rodent carcinogenicity of peroxisome proliferators and issues on human relevance.

A variety of substances such as hypolipidemic drugs, phthalate ester plasticizers, pesticides, and industrial solvents have been shown to increase the size and number of peroxisomes in rats and mice. They are grouped under the generic term peroxisome proliferators (PP) because of their unique property of inducing peroxisome proliferation. There are marked species differences in response to PP. Rats and mice are most sensitive, and hamsters show an intermediate response while guinea pigs, monkeys, and humans appear to be relatively insensitive or non-responsive at dose levels that produce a marked response in rodents. Out of over 100 PP identified to date, about 30 have been adequately tested and shown to be carcinogenic, inducing tumors (primarily in the liver) upon chronic administration to rats and/or mice; hence, chemicals which induce the proliferations of peroxisomes have formed a unique class of chemical carcinogens. It is not well documented that activation of the "peroxisome proliferator-activated receptor alpha" (PPARalpha) is involved in PP-induced liver growth and carcinogenesis in rodents. PPARalpha is also present in human cells; however, the levels reported are about 10% of those found in the liver of rodents. The human relevance of rodent tumors induced by PP has been the subject of debate over the last decade. Review of the existing evidence on PPAR-alpha agonists by a recent International Life Science Institute (ILSI) workgroup following a human relevance mode of action (MOA) framework has concluded that despite the presence of similar pathways in humans, it is unlikely that the proposed MOA for rodent tumors is plausible in humans, taking into account kinetic and dynamic factors. The data, however, did not permit a definitive conclusion that the animal MOA is not plausible in humans. While these agents appear unlikely to be hepatocarcinogens in humans at expected levels of human exposure, it remains uncertain to some experts in the field whether there is no possibility of carcinogenic potential under any circumstances of PP exposure, and if the potential human carcinogenicity of these chemicals can be summarily ignored. A number of remaining issues on human relevance of rodent tumors induced by PP are discussed.

Animals↗
PubMed2004

Selecting the appropriate rodent diet for endocrine disruptor research and testing studies.

Selecting the optimum diet for endocrine disruptor (ED) research and testing studies in rodents is critical because the diet may determine the sensitivity to detect or properly evaluate an ED compound. Dietary estrogens can profoundly influence many molecular and cellular event actions on estrogen receptors and estrogen-sensitive genes. The source, concentration, relative potency, and significance of dietary estrogens in rodent diets are reviewed, including dietary factors that focus specifically on total metabolizable energy and phytoestrogen content, which potentially affect ED studies in rodents. Research efforts to determine dietary factors in commercially available rodent diets that affect uterotrophic assays and the time of vaginal opening in immature CD-1 mice are summarized. A checklist is provided of important factors to consider when selecting diets for ED research and testing studies in rodents. Specific metabolizable energy levels are recommended for particular bioassays. Discussions include the between-batch variation in content of the phytoestrogens daidzein and genistein, the effects of total metabolizable energy and phytoestrogens on the timing (i.e., acceleration) of vaginal opening, and increased uterine weight in immature CD-1 mice. It is concluded that rodent diets differ significantly in estrogenic activity primarily due to the large variations in phytoestrogen content; therefore animal diets used in all ED studies should ideally be free of endocrine-modulating compounds.

Animal Feed↗
PubMed1996

Epidemiologic linkage of rodent and human hantavirus genomic sequences in case investigations of hantavirus pulmonary syndrome.

Sin Nombre virus (SNV) causes the zoonotic disease hantavirus pulmonary syndrome (HPS). Its mechanisms of transmission from rodent to human are poorly understood. It is possible that specific genetic signature sequences could be used to determine the probable site of each case-patient's exposure. Environmental assessments suggested 12 possible sites of rodent exposure for 6 HPS patients. Rodents were captured at 11 of the 12 sites and screened for SNV infection within 2 weeks of the patient's diagnosis. Viral sequences amplified from tissues of rodents at each site were compared with those from case-patients' tissues. Rodents bearing viruses with genetic sequence identity to case-patients' viruses across 2 genomic segments were identified in 4 investigations but never at >1 site. Indoor exposures to rodents were especially common at implicated sites. By distinguishing among multiple possible sites of exposure, viral genotyping studies can enhance understanding of the conditions associated with infection by SNV.

Animals↗
PubMed1999

Impact of microclimate on immature tick-rodent host interactions (Acari: Ixodidae): implications for parasite transmission.

Rodents play a significant role in enzootic cycles of tick-borne pathogens, notably, in the northern hemisphere, tick-borne encephalitis virus and Lyme borreliosis spirochaetes. The relative numbers of nymphal and larval ticks feeding on rodents are crucial variables in determining the probability of rodent infection and the degree of amplification of infection prevalence in the tick population. Manipulation of the microclimate within quasinatural experimental arenas revealed that under increasingly dry conditions the numbers of unfed nymphal Ixodes ricinus L. questing in upper layers of the herbage decreased, whereas the rate of fat use and the numbers of nymphs feeding on small rodents, both increased. This is consistent with nymphs descending to the moist lower vegetation layers for water replenishment, where they would come into contact with small hosts. Very few larvae quested or fed on rodents under the dry conditions, but many more did so once the humidity increased, suggesting that larvae escape desiccation by becoming quiescent. The ratio of larvae to nymphs feeding on rodents thus increases with increasing humidity, contributing to the seasonal and geographical variation in disease transmission dynamics.

Animals↗
PubMed1997

Some considerations for the development of diets for mature rodents used in long-term investigations.

Nutritional requirements for mature rodents used in long-term investigations are virtually unknown. The limited knowledge of the dietary needs of mature rodents is due in part to overreliance on weanling animals fed an experimental diet for relatively short periods. Generalizations made from observations of weanling rodents are not appropriate for all ages. Dietary recommendations for rodents have been established, for the most part, by using the nutritional benchmark of maximal growth rate in animals fed ad libitum. Although this method provides valuable insight into the understanding of nutritional deficiency, it is less effective in determining nutrient requirements for mature animals used for the long term. The implication that maximal growth resulting from ad libitum feeding may not indicate the best dietary regimen in the long term is consistent with the observation that energy-restricted rodents live significantly longer and have lower incidence of disease that do their ad libitum-fed counterparts. These and other findings discussed in the review suggest that nutrient requirements established for young rodents may need re-evaluation to determine their applicability to the dietary recommendations for older animals used in long-term investigations.

Animal Nutritional Physiological Phenomena↗
PubMed2005

Adaptive evolution of the insulin gene in caviomorph rodents.

Insulin is a conservative molecule among mammals, maintaining both its structure and function. Rodents that belong to the Suborder Hystricognathi represent an exception, having a very divergent molecule with unusual physiological properties. In this work, we analyzed the evolutionary pattern of the insulin gene in caviomorph rodents (South American hystricomorph rodents). We found that these rodents have higher rates of nonsynonymous:synonymous substitutions (d(N)/d(S)) than nonhystricomorph rodents and that values are heterogeneous inside the group. We estimated codons under positive selection, specifically the second binding site (A13 and B17) and others related with hexamerization (B18, B20, and B22). In the monomer structure, all selected sites formed a single patch around the second binding site. In the hexamer structure, these amino acids were grouped into three major patches. In this structure, contacts between B chains involved all selected sites (except B18), and between faces in the center of the molecule, all contacts were among selected sites. While there is no clear hypothesis regarding the cause of this drastic change, experimental evidence does show that this group of rodents has some peculiarities in growth function, and, whether coincidental or not, these changes appeared together with important changes in life-history traits.

Adaptation, Physiological↗
PubMed1997

Evolution of isochores in rodents.

The most deviant isochore pattern within mammals was found in rat and mouse; most other mammals possess a different kind of isochore organization called the "general pattern." However, isochore patterns remain largely unknown in rodents other than mouse and rat. To investigate the taxonomic distribution of isochore patterns in rodents, we sequenced the nuclear gene LCAT (lecithin:cholesterol acyltransferase) from 17 rodents species (bringing the total of LCAT sequences in rodent to 19) and compared their GC contents at third codon positions and in introns. We also analyzed an extensive sequence database from rodents other than rat and mouse. All murid LCAT sequences are much poorer in GC than all nonrodent LCAT sequences, and the hamster sequence database shows exactly the same isochore pattern as rat and mouse. Thus, all murids share the same special isochore pattern--GC homogenization. LCAT sequences are GC-poor in hystricomorphs too, but the guinea pig sequence database indicates that large changes in GC content occur without an overall modification of the isochore pattern. This novel mode of isochore evolution is called GC reordering. LCAT sequences also show that the evolution of isochores in sciurids and glirids is nonconservative in comparison with that in nonrodents. Thus, at least two novel patterns of isochore evolution were found. No rodent investigated to date shared the general mammalian pattern.

Animals↗
PubMed1994

Transmission of Rickettsia tsutsugamushi strains among humans, wild rodents, and trombiculid mites in an area of Japan in which tsutsugamushi disease is newly endemic.

Thirty-two newly isolated strains of Rickettsia tsutsugamushi, 14 from patients with tsutsugamushi disease, 12 from wild rodents, and 6 from trombiculid mites parasitizing rodents in Gifu Prefecture, Japan, were examined for reactivities to 12 monoclonal antibodies by an indirect fluorescent-antibody test to classify their antigenicities. All of the isolates could be classified into one of six groups (KN-1, KN-2, KN-3, GJ-1, R158, and R161) according to their reactivities to the monoclonal antibodies. The KN-1 and GJ-1 strains that are prevalent among patients from Gifu Prefecture had the same reactivities as the Kawasaki and Kuroki strains, respectively, which have been isolated and are prevalent in the Miyazaki and Kagoshima prefectures in southwest Japan. The isolates from patients were different in serotype from those from rodents and mites (Leptotrombidium pallidum). The KN-2 and KN-3 strains were most prevalent among patients and among rodents and mites, respectively. No close similarity between KN-2 and other strains tested was observed. KN-3 is only a minor contributor to diseases in patients in Gifu Prefecture; however, it was proven that the same strain was prevalent in Niigata Prefecture in northern Japan. Thus, Gifu Prefecture is an area where southern, northern, and local strains are found. We hypothesize that humans are prone to infection with KN-2, GJ-1 (very similar to Kuroki), and KN-1 (very similar to Kawasaki), probably by infestation with Leptotrombidium scutellare. While both L. scutellare and L. pallidum parasitize wild rodents and may carry any rickettsial strain, the most virulent strain, KN-3, is predominant among wild rodents. Antigenic analysis using monoclonal antibodies to R. tsutsugamushi should be useful for epidemiological studies of infection with this organism.

Animals↗
PubMed2005

Commercial rodent diets contain more sodium than rats need.

The dietary sodium requirements for rats have been a matter of debate. Our hypothesis was that normal commercial rodent chow contains sodium in excess of dietary needs and that this could have a significant impact on cardiovascular and renal physiology. To investigate dietary sodium requirements, 3-wk-old weanling Sprague-Dawley rats were fed a custom pelleted diet containing no sodium that was isocaloric to normal commercial rodent chow. These rats were provided with two drinking bottles; one contained water, and the other contained 0.5% NaCl. Thus they could choose and consume sodium as needed. Age-matched controls received normal pelleted Harlan Teklad 22/5 rodent diet (0.5% sodium content) and water ad libitum. Body weight and liquid intake were monitored over 7 wk until the rats were 10 wk old. At the end of the study, blood pressure was recorded. Weekly sodium intake in the experimental group was only 15% of that reported for rats fed normal rodent chow beginning in the first week postweaning. Growth was identical in the two groups (7.8 +/- 0.1 vs. 7.6 +/- 0.1 g/day), as was the total fluid volume intake. Blood pressure was significantly lower in the experimental rats compared with controls (96 +/- 4 vs. 122 +/- 4 mmHg, P < 0.05). These data suggest that, when given the choice, rats will consume significantly less sodium than provided in commercial chow, without any alteration in their growth rate. Rats fed standard commercial rodent chow may consume at least seven times more sodium than is necessary. This suggests commercial rodent diets may force excess sodium to accommodate caloric intake.

Animal Feed↗
PubMedDate not supplied

Rodent carcinogenicity bioassay: past, present, and future.

Toxicity/carcinogenicity studies in rodents have played a pivotal role in identifying chemicals that are potentially hazardous to humans. In fact, nearly all of the known human carcinogens are also carcinogenic in 1 or more rodent species. During the past 20 yr the quality and consistency of rodent studies has improved considerably, and much has been learned about mechanisms whereby chemicals initiate or promote the carcinogenic process in rats and mice. The process of identifying chemicals that cause toxicity or carcinogenicity in rodents is quite well established, but the procedures for extrapolating this data for risk management decisions in the protection of human health have lagged far behind. While many would accept the assumptions that genotoxic chemicals that cause cancer in animals pose a cancer risk to humans and that genotoxic chemicals causing cancer at high doses pose a risk at lower doses, there is much less certainty with respect to nongenotoxic chemicals. The confusion about risk extrapolation for nongenotoxic chemicals has often lead to criticism of the hazard identification process for chemicals in general. There is increasing awareness of the complexity of the carcinogenic process that has made species extrapolation and dose extrapolation from rodent studies to humans more complex. Although newer molecular biological techniques and cell kinetic measurements offer exciting possibilities for better risk assessment, it is the combination of well-designed rodent studies with appropriate mechanistic studies that offers the best hope for regulatory decisions based on sound scientific principles.

Animals↗
PubMed2002

The migration and localization of Loa loa infective and fourth-stage larvae in normal and immunosuppressed rodents.

The migration and localization of the human filarial parasite Loa loa in laboratory mice (BALB/c and Swiss) and jirds (Meriones unguiculatus) was investigated. The rodents, either left immunocompetent or immunosuppressed with hydrocortisone, were each inoculated subcutaneously or intraperitoneally with 50 or 200 infective, third-stage larvae (L(3)) of L. loa. Groups of the rodents were killed at various times post-infection, up to day 40, to enable histological studies and permit developing larvae to be recovered. Larvae survived and developed for only 1 week in the immunocompetent rodents but for a mean of 3 weeks in the immunosuppressed. Most of the larvae were found in the subcutaneous tissues (81.9%), peritoneal cavity (14.9%), pleural cavity (1.8%) or the lungs and heart (1.3%) and none was detected in the spleen, kidney, intestine, liver or pancreas. Localization of the larvae appeared unaffected by the site of inoculation, the rodent species or strain, or the dose of L(3) used. The recovery of larvae (as a percentage of the number inoculated) was better among the rodents inoculated with 50 L(3) each than among those given four times as many L(3). The results of the histological studies not only confirmed the presence of larvae in the subcutaneous tissue (72.5%), muscles (11.7%) and peritoneal and pleural cavities (7.8%) of the infected rodents but also revealed worms in the lymphatic vessels of the mesentery and spinal cord (7.3%). These results indicate that most L. loa L(3) inoculated into a mammalian host localize in the cutaneous sites and that only a small proportion of them might migrate, using the lymphatic system, into the internal organs. The observation of migrating L. loa larvae in the lymphatic vessel of the meningeal envelope of the spinal cord, albeit in an experimental host, may explain why, in areas where human loiasis is endemic, neurological manifestations occasionally occur in those with L. loa infections.

Animals↗
PubMed2005

Conservation of pregnancy-specific glycoprotein (PSG) N domains following independent expansions of the gene families in rodents and primates.

BACKGROUND: Rodent and primate pregnancy-specific glycoprotein (PSG) gene families have expanded independently from a common ancestor and are expressed virtually exclusively in placental trophoblasts. However, within each species, it is unknown whether multiple paralogs have been selected for diversification of function, or for increased dosage of monofunctional PSG. We analysed the evolution of the mouse PSG sequences, and compared them to rat, human and baboon PSGs to attempt to understand the evolution of this complex gene family. RESULTS: Phylogenetic tree analyses indicate that the primate N domains and the rodent N1 domains exhibit a higher degree of conservation than that observed in a comparison of the mouse N1 and N2 domains, or mouse N1 and N3 domains. Compared to human and baboon PSG N domain exons, mouse and rat PSG N domain exons have undergone less sequence homogenisation. The high non-synonymous substitution rates observed in the CFG face of the mouse N1 domain, within a context of overall conservation, suggests divergence of function of mouse PSGs. The rat PSG family appears to have undergone less expansion than the mouse, exhibits lower divergence rates and increased sequence homogenisation in the CFG face of the N1 domain. In contrast to most primate PSG N domains, rodent PSG N1 domains do not contain an RGD tri-peptide motif, but do contain RGD-like sequences, which are not conserved in rodent N2 and N3 domains. CONCLUSION: Relative conservation of primate N domains and rodent N1 domains suggests that, despite independent gene family expansions and structural diversification, mouse and human PSGs retain conserved functions. Human PSG gene family expansion and homogenisation suggests that evolution occurred in a concerted manner that maintains similar functions of PSGs, whilst increasing gene dosage of the family as a whole. In the mouse, gene family expansion, coupled with local diversification of the CFG face, suggests selection both for increased gene dosage and diversification of function. Partial conservation of RGD and RGD-like tri-peptides in primate and rodent N and N1 domains, respectively, supports a role for these motifs in PSG function.

Amino Acid Motifs↗
PubMed1999

Environmental enrichment for laboratory rodents: animal welfare and the methods of science.

Because of the difficulty of' establishing objective measures of laboratory rodents' psychological well-being, developing environmental enrichment programs that are actually beneficial to rodents destined to participate in laboratory research is particularly challenging. Many studies of effects of environmental complexity, social housing, and increases in cage size suggest that professional judgments as to the impact of diverse types of environmental enrichment on rodent welfare are not a reliable basis for evaluating the outcomes of enrichment programs for laboratory rodents. Successful enrichment programs will vary from one rodent species to another, between sexes, as well as between age classes. There is a need for objective, measurable goals for proposed environmental enrichment programs for rodents, as well a s for empirical investigations of the beneficial and detrimental consequences of proposed environmental manipulations.

Journal Article↗
PubMed2006

Isoflurane leakage from non-rebreathing rodent anaesthesia circuits: comparison of emissions from conventional and modified ports.

Chronic exposure to low levels of fluorocarbon-based waste anaesthetic gas (WAG) has been linked to a multitude of human health problems. We have shown that isoflurane exhaust from passive gas-scavenging canisters is often quite high when using conventional rodent anaesthesia protocols and equipment. Another likely source of WAG build-up in rodent procedure rooms is leakage at the interface between the breathing circuit and the animal's face. We evaluated this possibility using three non-rebreathing circuits: traditional Bain, modified Bain, and Mapleson (type E). For the Mapleson E circuit, a conical rodent facemask was attached and used in one of two configurations: normal aperture, or aperture modified with a latex diaphragm (cut from an unpowdered surgical glove) to reduce the orifice diameter and tighten the seal. Adult female Sprague-Dawley rats were anaesthetized with isoflurane (5% for induction, 2% or 3.5% for maintenance) in oxygen (2 L/min for induction, 1 L/min for maintenance). Isoflurane leakage was assessed by real-time spectrophotometry. In 94% of the trials, three configurations - traditional Bain, modified Bain, and Mapleson E with unmodified mask - permitted isoflurane leakage approaching or exceeding 100 ppm at the face/port interface. In contrast, the Mapleson circuit with diaphragm-modified mask emitted significantly (P<or=0.0003) less isoflurane (peak of 9.5+/-1.7 ppm [mean+/-standard error]). These data indicate that (1) WAG leakage from standard rodent non-rebreathing circuits is substantial, and that (2) a simple, rapid, and economical modification to a conventional rodent facemask can significantly reduce WAG exposure to workers performing many rodent anaesthesia procedures in one session.

Air Pollutants, Occupational↗