The urinary bladder carcinogen propoxur does not produce genotoxic effects in the urinary bladder of Wistar male rats.

Propoxur (PPX) is a carbamate insecticide which induced urinary bladder cancer in Wistar rats when fed at 5000ppm in Altromin 1321 diet (1321). In the present investigation, PPX was studied for induction of several key events related to modes of action (MOA) of carcinogenicity in urinary bladders (UBs). Wistar rats were administered the compound for 28 days at 8000ppm in Provini Liba SA 3883 diet, which is similar to the 1321 diet. o-Anisidine HCl (AH) was used as a genotoxic UB carcinogenic comparator, and trisodium nitrilotriacetate (NTA) as an epigenetic UB carcinogen comparator. Along with the non-dosed control and three test substance groups (PPX, AH, NTA), four more groups were additionally fed 2% ammonium chloride (AC) in the diet to acidify the urine, since 1321 was reported to increase urinary pH. AC did acidify the urine, as expected, although the 3883 diet itself did not increase pH values above 8. In the alkaline comet assay, AH produced DNA single strand breaks (SSBs) in the UB urothelium (UBU) irrespective of AC administration, whereas PPX and NTA did not. In the nucleotide (32)P-postlabeling assay (NPL), AH produced DNA adducts irrespective of AC administration, whereas PPX and NTA did not. Routine (H&E) histopathology evaluation of the UBU did not reveal any hyperplasia or evidence of luminal microprecipitates or calculi in any of the groups. Assessment of UBU proliferation as measured by immunohistochemistry of proliferating cell nuclear antigen, revealed that NTA and NTA plus AC increased the replicating fraction (RF). Also AH plus AC, but not AH alone, increased the RF of UBU, whereas PPX groups were not significantly different from controls. Thus, the results reveal no evidence for DNA SSBs, binding, or alteration of DNA synthesis in the UBU by PPX, while demonstrating UBU DNA damage by AH and showing that NTA does not damage DNA, but causes increased UBU proliferation. The findings are in accord with a genotoxic MOA for AH, and an epigenetic MOA for NTA. The MOA of PPX does not involve genotoxicity and may be specific to the 1321 diet.

Developmental and reproductive toxicity studies on artemisone.

BACKGROUND: In order to justify clinical studies in women of child-bearing age with artemisone, a new artimisinin derivative, studies to assess fertility and early embryonic development in rats, developmental toxicity in rats and rabbits, and peri-post natal development in rats were performed. METHODS AND RESULTS: In the study on fertility and early embryonic development (dose levels 0-5-20-80 mg/kg bw/day), doses inducing clinical and organ toxicity were used. Only in severe toxicity conditions, a reduction of the number of estruses, a prolonged time to insemination, decreased numbers of corpora lutea, implantation sites, and viable fetuses were found. Two developmental toxicity studies were performed in rats (dose levels 0-1-2 mg/kg bw/day) and rabbits (dose levels 0-2.5-5.0-7.5 mg/kg bw/day). It was shown that rats were about 5 times more sensitive than rabbits. In rats, artemisone induced total litter loss (late resorptions) at 2 mg/kg body weight and above with an increased incidence of a common vascular variation and retarded ossification at this dose. In rabbits, maternal toxicity, abortion and a slightly increased incidence of cardiac ventricular septal defects was observed at 7.5 mg/kg body weight. In a pre- and postnatal developmental toxicity study in rats (dose levels 0-1-2-4 mg/kg bw/day), 4 mg/kg body weight artemisone induced clinical symptoms and affected postnatal survival, body weight gain in the F1 pups, and motor activity. CONCLUSIONS: In summary, artemisone was shown to be embryo- and fetotoxic and induced cardiac ventricular septal defects and retarded ossification in dosages where total litter loss and abortions were observed. However, no effect on reproductive and developmental parameters below severe toxic dosages could be observed.

Effectiveness of workplace rehabilitation interventions in the treatment of work-related low back pain: a systematic review.

PURPOSE: A systematic review was conducted to evaluate the effectiveness of workplace rehabilitation interventions for injured workers with low back pain (LBP). METHOD: MEDLINE, CINAHL (Cumulative Index to Nursing and Allied Health Literature), EMBASE, and AMED (Allied and Complementary Medicine) were searched from 1982 to 2005 for peer-reviewed studies of rehabilitation interventions that were provided at the workplace to workers with musculoskeletal work-related LBP. Methodological quality appraisal and data extraction were conducted by five reviewers. RESULTS: Of a total of 1,224 articles that were identified by the search, 15 articles, consisting of 10 studies, were of sufficient quality to be included in the review. The best evidence was that clinical interventions with occupational interventions as well as early return to work/modified work interventions were effective in returning workers to work faster, reducing pain and disability, and decreasing the rate of back injuries. Ergonomic interventions also were found to be effective workplace interventions. CONCLUSION: The need for further research in this area is necessary to reduce the burden of back pain on employees and their families, employers, and the health care system.

c-fos mRNA expression in rat cortical neurons during glutamate-mediated excitotoxicity.

We have previously reported that exposure of mouse cerebellar granule cells (mCGCs) to excitotoxic concentrations of glutamate (Glu) induced a delayed, elevated, and sustained expression of c-fos mRNA, which was N-methyl-D-aspartic acid (NMDA) receptor mediated. In this study, the overstimulation of Glu receptors in primary rat cortical neurons by excitotoxins was used to study the cellular events triggering excitotoxic neuronal cell death, as the rat is the preferred species in regulatory and nonregulatory toxicological investigations. Exposure of rat cortical neurons to excitotoxins at high, toxic concentrations showed a change in the c-fos mRNA expression profile from a transient expression to one of sustained elevated levels. The excitotoxins induced much higher levels of c-fos mRNA in rat cortical neurons than in the mouse CGC system. Glu-induced c-fos mRNA expression, under excitotoxic conditions, was inhibited by D-2-amino-5-phosphonopentanoate (AP5) but not 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), indicating an event mediated by the NMDA subtype of Glu receptors. Using 12 compounds, which covered a range of nontoxic, toxic, and excitotoxic effects on rat cortical neurons, excitotoxicity was paralleled by a sustained, elevated c-fos mRNA expression. Furthermore, on account of the high expression levels of c-fos mRNA under excitotoxic conditions, it is suggested that an unambiguous elevation in c-fos mRNA expression at a single time point of 60 min can be used to predict the excitotoxic properties of a range of functionally different chemical compounds. In view of the high levels of expression of c-fos mRNA, the rat cortical cell system may also be used as a more sensitive model than mCGCs for investigations into early markers of excitotoxicity.

Oxidative stress in rat cortical neurons and astrocytes induced by paraquat in vitro.

Oxidative stress has been discussed as crucial mechanism of neuronal cell death in the adult brain. However, it was not clear until now whether neurons are more sensitive to oxidative stress than the other cells in the brain, e.g. astrocytes. Therefore both cell types were exposed to oxidative stress provoked by the redox-cycling compound paraquat. Cortical neurons were found to be more sensitive towards paraquat toxicity than astrocytes as shown by MTT and Neutral Red assay, two different cytotoxicity assays. Mitochondrial functions were determined by the mitochondrial membrane potential and intracellular ATP concentrations. Again cortical neurons were more severely impaired (by paraquat than astrocytes). The production of reactive oxygen species after paraquat exposure was much higher in cortical neurons than in astrocytes and correlated with a higher depletion of GSH (intracellular glutathion). Lipid peroxidation could be shown in astrocytes via the breakdown product malondialdehyde (MDA) whereas in cortical neurons 4-hydroxynonenal (4-HNE) was detected as this endpoint. If and how oxidative stress influences the antioxidant defense was determined via changes in the expression of antioxidant enzymes. Paraquat exposure lead to a 2-3 fold increase of catalase, MnSOD and CuZnSOD mRNA expression in astrocytes. In contrast to astrocytes, in cortical neurons catalase and MnSOD mRNA levels were only marginally elevated above 1.5-fold by treatment with paraquat. Expression levels of glutathione peroxidase (GPx) mRNA were the only one that were not changed in both cell types after paraquat exposure. It is concluded that the more marked increase in expression levels of antioxidant enzymes may render astrocytes more resistant to oxidative stress than neuronal cells.

Changes in antioxidant enzyme expression in response to hydrogen peroxide in rat astroglial cells.

Oxidative stress has been causally linked to a variety of neurodegenerative diseases. To clarify the role of the antioxidant enzyme (AOE) system in oxidative brain damage primary cultures of rat astroglial cells were exposed to hydrogen peroxide (H2O2). Expression of AOEs and several parameters for cell viability and functionality were measured. In our experiments astrocytes responded to low concentrations of H2O2 exposure with a pronounced generation of ROS which ran parallel with induction of lipid peroxidation. This distinct oxidative stress was not reflected in cell viability or functionality parameters measured. Cytotoxicity, a decrease in glutathione content of astrocytes, and impairment of mitochondrial functions became obvious only for higher concentrations of H2O2. After H2O2 exposure catalase, manganese superoxide dismutase, and glutathione peroxidase expression levels were found to be increased, whereas copper/zinc superoxide dismutase mRNA expression was not affected. These data indicate that the AOE system of astrocytes can be directly regulated by oxidative stress and may thus contribute to protection of cells against oxidative insults.

The influence of oxidative stress on catalase and MnSOD gene transcription in astrocytes.

The brain is particularly vulnerable to oxygen free radicals, which have been implicated in the pathology of several neurological disorders. The antioxidant enzyme (AOE) system of the brain may play an important role in the protection against such oxidative stress. We investigated the influence of oxidative stress on the transcription of catalase and MnSOD mRNA. Primary rat astroglial cell cultures were treated either with hydrogen peroxide (H2O2), as a direct mediator of oxidative stress, or with the redox cycling compound paraquat. Both substances led to an increase of catalase and MnSOD mRNA levels. To further elucidate the mechanisms residing behind this increase, transfection experiments were performed. Transient transfection of primary astroglial cells with a reporter plasmid containing the upstream region of the catalase gene showed a decrease in reporter gene activity after exposure of transfected cells to either H2O2 or paraquat. In contrast, transfection experiments done with reporter plasmids for the MnSOD upstream region resulted in an increase of reporter gene activity after H2O2 as well as after paraquat treatment of transfected cells. These results indicate transcriptional regulation of MnSOD and post-transcriptional regulation of catalase gene expression after oxidative stress in primary rat astrocytes.

[The importance of standardised cell culture methods for the routine toxicology in pharmaceutical companies]. / Die Bedeutung standardisierter Zellkulturverfahren für die Routine-Toxikologie der Pharmaindustrie.

The establishment and the use of standardised cell cultures build the basis for toxicological investigations and guarantees the comparison over years. The requirement for these investigations in vitro are mainly primary cell cultures from the target organs. The cell cultures should keep, also under long term culture conditions, the organ differentiation. This will be supported by new developments in the fields of cell culture media, supplements and coating material of the cell culture dishes. Routinely, cells from toxicologically important organs like liver, kidney and nervous system were used in vitro. However, mechanistic investigations of toxicological findings in vivo made the use of cell systems from other organs like cartilage, bone, skeletal and heart muscle cells, testis eg necessary. All cell culture systems were established and standardised to allow repeated tests under the same conditions. The determination of characteristic proteins or enzymes of the related organ will demonstrate the organ like quality of the cell cultures. The central question of toxicology is the risk assessment. Here, the in vitro toxicology will provide important information by a comparison between human and animal cell cultures under the same conditions.

Rat cortical neuron cultures: an in vitro model for differentiating mechanisms of chemically induced neurotoxicity.

Various structurally unrelated chemicals [2,5 hexandione, acrylamide, organophosphates like mipafox, beta,beta iminodipropionnitrile (IDPN), 3-nitropropionic acid (3-NP), potassium cyanide (KCN), paraquat, and NMDA (N-methyl-D-apartic acid)] are known to cause degenerative damage of the peripheral or central nervous system. Differentiated neuronal cell cultures obtained from fetal rats have been used to differentiate the mechanisms underlying this type of neurotoxicity. Cytotoxicity as measured by a viability assay was not sensitive enough and had to be supplemented by further endpoints covering effects on cytoskeleton and on the energy state of the cells [glucose consumption, mitochondrial membrane potential and adenosine 5'-triphosphate (ATP) concentration]. Compounds like the delayed neurotoxic organophosphates, exert a selective direct effect on cytoskeleton elements in this model at concentrations distinctly below cytotoxic concentrations. Other compounds, like KCN, paraquat, and 3-NP selectively disrupt the balance between energy supply and demand of the neurons either by interacting with mitochondrial respiration or glycolysis. For these compounds cytoskeletal damage seemed to be secondary to the energy depletion. For NMDA, 2,5 hexandione and acrylamide, both mechanisms may contribute to the neuronal damage. In conclusion, primary cortical neuronal cultures of the rat are well suited to detect a neurotoxic potential and to differentiate its underlying mechanisms. Damage of the cytoskeleton may be considered as an endpoint mechanistically related to degenerative neuropathic effects.

Establishment of an in vitro screening model for neurodegeneration induced by antimalarial drugs of the artemisinin-type..

The establishment of an in vitro screening model for neurodegeneration inducing antimalarial drugs was conducted in stepwise fashion. Firstly, the in vivo selective neurotoxic potency of artemisinin was tested in neuronal cells in vitro in relation to the cytotoxic potency in other organ cell cultures such as liver and kidney or versus glial cells. Secondly, a comparison between different parts of the brain (cortex vs. brain stem) was performed and in the last step, a fast and sensitive screening endpoint was identified. In summary, non-neuronal cell lines such as hepatocytes (HEP-G2), liver epithelial cells (IAR), proximal tubular cells (LLC-PK(1)) and glial cells from the rat (C6) and human (GO-G-IJKT) displayed only moderate sensitivity to artemisinin and its derivatives. The same was found in undifferentiated neuronal cell lines from the mouse (N-18) and from human (Kelly), whereas during differentiation, these cells became much more sensitive. Primary astrocytes from the rat also were not specifically involved. In the comparison of primary neuronal cell cultures from the cortex and brain stem of the rat, the brain stem was found to be more sensitive than the cortex. The neurotoxic potential was determined by cytoskeleton elements (neurofilaments), which were degradated in vitro by diverse neurodegenerative compounds. In comparison of dog and rat primary brain stem cultures, the dog cells were found to be more sensitive to artemisinin than the rat cells. In addition to the primary brain stem cell cultures it was shown that the sprouting assay, which determines persistent delayed neurotoxic effects, is also useful for screening antimalarial drugs. To other compounds, artemether and artesunate, showed that use of the sprouting assay followed by primary brain stem cultures of the rat will be a good strategy to select candidate compounds.

Determination of the excitatory potencies of fluoroquinolones in the central nervous system by an in vitro model.

Fluoroquinolones have been reported to induce central nervous system side effects, including seizures and psychiatric events. Although relatively rare in patients up to now, the proconvulsant activity depends on the chemical structure and might be a critical endpoint of some new representatives of this valuable class of antimicrobials. The electrophysiological determination of field potentials in the CA1 region of the rat hippocampus slice allowed an assessment of the excitatory potential of fluoroquinolones and might be predictive for their neurotoxic potency in vivo. An optimization of this method and its extension to other fluoroquinolones resulted in a defined rank order. Well-known already-marketed quinolones as well as some fluoroquinolones under evaluation and development were used. The dose range tested was between 0.5 and 4 mumol/liter, which was comparable to the therapeutic concentration in the brain. All tested compounds increased the population spike amplitude in a concentration-dependent manner, and the resulting excitatory potency was highly dependent on the chemical structure, with compounds ranging from least to most excitatory as follows: ofloxacin, ciprofloxacin, nalidixic acid, moxifloxacin, BAY x 8843, [corrected] fleroxacin, lomefloxacin, enoxacin, clinafloxacin (much more excitatory than enoxacin), tosufloxacin, trovafloxacin, BAY 15-7828, and BAY x 9181 (much more excitatory than BAY 15-7828). The proposed hippocampus slice model not only is suitable for giving valuable alerts as to convulsive potential during candidate selection but also enables mechanistic investigations. These investigations pointed to the N-methyl-D-aspartate receptor as the probable target of the fluoroquinolone effects.

Learning and memory of rats after long-term administration of low doses of parathion.

A set of four learning and memory tests (Morris Maze I for reference memory, Morris Maze II for working memory, one-way active avoidance, and passive avoidance) were employed to address the questions whether parathion impaired cognitive functions after low, long-term exposure and could cause persistent changes in cognition. Motor activity and general behavior were investigated in a functional observational battery. Parathion was administered in rat food in low doses which caused no clinical symptoms and no or borderline brain acetylcholinesterase inhibition. Parathion doses of 0.5, 2, or 8 ppm in rat food produced the averaged uptake of 24, 100, or 400 microg/kg body weight per group per day in male rats and 36, 152, or 550 microg/kg per day in female rats in week 13. Learning tests were performed in weeks 1 to 4 and 10 to 14, as well as 30 to 34 weeks after the end of treatment, when the male and female rats were about 13 months old. Low doses of parathion given daily for 13 weeks had no cumulative or adverse effects on learning and memory, either during treatment or after the extended treatment-free period, in any of the tests. A significant improvement of learning compared to control observed in the Morris Water Maze I during the first week of treatment (males dose group 0.5 ppm) shows that parathion can improved cognitive functions in rats. Results of the study indicate that adverse effects changing learning and memory in animals may occur only at higher doses of organophosphates, at which the peripheral and brain acetylcholinesterases are inhibited to a greater extent than those in the present study.

Improved in vitro method for screening organophosphate-induced delayed polyneuropathy.

Organophosphate-induced delayed polyneuropathy (OPIDP) has been tested mainly byin vivo methods in the chicken and by examination of the inhibition of neuropathy target esterase in their neuronal tissue. An alternative method, using permanent neuronal cell lines and detecting the growth of neurite-like sprouts by morphometric means, has meanwhile been validated to substitute thesein vivo investigations, at least in part. This paper reports some modifications of this in vitro method in order to optimize it for a routine laboratory screening test of organophosphates. By determination of cytoskeletal elements with cell ELISA, it was demonstrated that seven OPIDP-inducing compounds [tri-o-cresyl phosphate (TOCP) and its metabolite cresyl-saligeninphosphate, haloxon, mipafox, leptophos, EPN and chlorpyrifos] and nine negative control substances [paraoxon, methylazinphos, ethylazinphos, dimefox, dimethoate, phenylmethanesulfonylfluoride (PMSF) and three organophosphate metabolite-cresol, phenol and p-nitrophenol] resulted in the same effects as seen in the original test method. The cell ELISA technique thus represents an alternative method that is much easier to perform than the morphometric method.

An in vitro model for toxicological investigations of environmental neurotoxins in primary neuronal cell cultures.

Currently, most neurotoxicological investigations are still conducted using various animal models (e.g. chickens, rodents). In this report, alternative strategies of testing were examined to detect the neurotoxic potency of foreign compounds. Primary neuronal cell cultures from fetal rats are already an accepted model for mechanistic and pharmacological studies in drug research. Their suitability for neurotoxicological studies was examined by using industrial model compounds, which are well-known inductors of neuropathies: acrylamide, hexachlorophene, paraquat, n-hexane, and its neurotoxic metabolites acetylaceton and 2,5-hexandione. As a control compound, the nonneurotoxic solvent n-heptane was used. General cytotoxicity and the intracellular content of glial fibrillary acid protein, neuron-specific enolase, and neurofilaments were measured. n-Heptane induced an acute cytotoxicity and acrylamide and 2,5-hexandione produced a delayed cytotoxicity in primary neuronal cells, whereas the others showed no cytotoxic potency in the tested concentration range. These results were in agreement with the quantification of neurons by neuron-specific enolase. In contrast, with the exception of acetylaceton, glia cells were significantly affected by all neurotoxins at the later time. Signs of axonopathies were demonstrated for acrylamide, n-hexane and its metabolites, as well as for hexachlorophene and paraquat in vitro, by determining the intracellular neurofilament level. Therefore, the determination of cell-specific end points is necessary to detect the neurotoxic potency and quality of a compound, whereas the cytotoxicity assay limited the tested concentration range.

Genotoxicity of neurotoxic triaryl phosphates: identification of DNA adducts of the ultimate metabolites, saligenin phosphates.

2-Phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide is an electrophilic and neurotoxic metabolite of o-tolyl phosphates. We have investigated the genotoxicity of this saligenin phosphate and the structure of adducts formed by incubation of 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide with nucleosides and DNA. o-Tolyl phosphate was mutagenic in the Ames test (695 revertants/mumol, Salmonella typhimurium TA 100) only with metabolic activation. 2-Phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide, which is a cyclization product similar to those expected from o-tolyl phosphate, was a potent mutagen in bacteria (1452 revertants/mumol, S. typhimurium TA 100) which did not require metabolic activation. Incubation of 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide with guanosine, deoxycytidine, and deoxyadenosine resulted in formation of guanosine, deoxyuridine, and adenine adducts. These were identified as N-2-(o-hydroxybenzyl)guanosine, N-3-(o-hydroxybenzyl)deoxyuridine, N-1-(o-hydroxybenzyl)adenine, and N-3-(o-hydroxybenzyl)adenine by 1H-NMR spectroscopy, thermospray mass spectrometry, and pH-dependent electronic spectrometry. The deoxyuridine adduct is formed by an alkylation at N-3 of deoxycytidine followed by conversion of the adjacent exocyclic imino group to carbonyl (hydrolytic deamination). The formation of N-2-(o-hydroxybenzyl)-deoxyguanosine, N-3-(o-hydroxybenzyl)deoxyuridine, and N-1-(o-hydroxybenzyl)deoxyadenosine was also demonstrated when 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide was incubated with calf thymus DNA. Adducts formed with nucleosides in calf thymus DNA reacted with 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide in vitro were detected by the 32P-postlabeling technique and identified by comparison with synthetic references. DNA adducts are formed by an o-hydroxybenzylation from cyclic phosphoranes derived from o-alkyl-substituted triaryl phosphates.(ABSTRACT TRUNCATED AT 250 WORDS)

Chlorophyll fluorescence lifetime determination of waterstressed C3- and C4-plants.

This article describes the effect of water stress on the room temperature chlorophyll fluorescence lifetime of plants of wheat (C3) and maize (C4). In addition, net CO2 assimilation rate (PN), stomatal conductance and the fluorescence quenching coefficients qP and qNP at steady state conditions were recorded. The overall fluorescence decay of the control plants can be described by an average decay time of 1 ns for both plant types. Water stress did not modify this parameter in the case of wheat, whereas a shortening of the decay was observed for waterstressed maize plants (tau = 0.45 ns). This shortening in the chlorophyll fluorescence decay was accompanied by an increase in the non-photochemical quenching (qNP). The photochemical quenching (qP) and therefore the electron transport via photosystem II remains unaffected by water stress. The most pronounced effect of the stress for both plant types was a decrease in PN due to a closure of the stomata.

The inhibitory effect of neuropathic organophosphate esters on neurite outgrowth in cell cultures: A basis for screening for delayed neurotoxicity.

Organophosphates have previously been tested for the induction of delayed neuropathy in adult hens. An alternative in vitro test, which avoids the severe suffering caused by the test in hens, has been developed using permanent cell lines from a rat-brain glioma (C-6) or from a mouse-brain neuroblastoma (N-18). Addition of dibutyryl cAMP to these cell cultures triggers the development of neurite-like processes; the development of these processes is inhibited by the addition of various organophosphate compounds and this inhibition serves as an indicator of neurotoxicity. 26 compounds with positive results in the in vivo test in hens, and eight analogues with negative results were tested in vitro. An almost perfect correlation between the in vivo and in vitro results was found; two compounds with weak positive results in vitro (O,O,S-trimethyl phosphorothioate and O,S,S-trimethyl phosphorothioate) yielded negative results in hens but this discrepancy may be related to their bioavailability in hens. The in vitro test is recommended to avoid the pain and stress caused by testing in vivo in hens.

Time-resolved spectroscopy of the blue fluorescence of spinach leaves.

When excited by ultraviolet radiation, leaves of a great number of species of higher plants exhibit emission of blue fluorescence, comparable in intensity to the red emission of chlorophyll. The fluorescence decay of the blue emission of spinach leaves recorded by single photon counting techniques is decomposed into exponential components and it is shown that at least three different components are present. The lifetime of the three components does not show significant variations with the excitation or emission wavelengths. The excitation and emission spectra of each component were determined. The nature of the chemical compounds which cause this emission is discussed in relation to these spectra.