Neurogenic cutaneous vasodilatation and plasma extravasation in diabetic rats: effect of insulin and nerve growth factor.

1. Neurogenic vasoactive responses in rat skin were investigated following 8 weeks of streptozotocin-induced diabetes to determine the effect of diabetes and of treatment with insulin and nerve growth factor (NGF) treatment. 2. Diabetic rats were divided into three groups: untreated; insulin (4 IU day(-1) by s.c. implant weeks 4-8) treated; Nerve Growth Factor, NGF, (0.2 mg kg(-1) three times weekly, weeks 4-8) treated. A fourth group served as a non-diabetic control. 3. Electrical stimulation of the saphenous nerve (10 V, 2 Hz, 1 ms for 30 s) increased blood flow in the ipsilateral paw skin, as measured by laser Doppler flowmetry. The peak increase was similar between groups, but the time taken for flow to return to a steady baseline was significantly (P < 0.01) reduced in untreated diabetic rats, when compared with non-diabetic controls, but not significantly reduced in the insulin- or NGF-treated diabetic groups. 4. A second stimulation of the saphenous nerve (10 V, 2 Hz, 1 ms for 5 min) produced plasma extravasation, measured by the extravascular accumulation of 125I-albumin, in the skin. Plasma extravasation was significantly attenuated (P < 0.001) in the untreated diabetic group, but not the insulin-treated group, compared to non-diabetic controls. Plasma extravasation was present, though reduced, in the NGF-treated group. 5. Plasma extravasation induced by intradermal injections of substance P with and without CGRP was similar in all groups indicating no decrease in vascular responsiveness to exogenously applied neuropeptides. The results suggest that release of neuropeptides is diminished in diabetes and that treatment with either insulin or NGF can restore neurogenic microvascular vasoactive responses towards normal.

Nerve growth factor- and neurotrophin-3-induced changes in nociceptive threshold and the release of substance P from the rat isolated spinal cord.

Acute superfusion of nerve growth factor (NGF; 1-100 ng/ml) through a naive rat spinal cord preparation did not alter basal or electrically evoked release of substance P-like immunoreactivity (SP-LI). In contrast, neurotrophin-3 (NT-3; 1-100 ng/ml), although not modifying SP-LI basal outflow, dose-dependently inhibited the electrically evoked, but not capsaicin (10 nM)-induced, release of the peptide. This NT-3 (10 ng/ml)-induced inhibition persisted even in the presence of 100 ng/ml NGF in the perfusion fluid and was still significant when the evoked release of SP-LI was enhanced by a prolonged in vivo treatment with NGF. Co-superfusion with naloxone (0.1 microM), but not CGP 36742 (100 microM), a GABAB antagonist, prevented NT-3 (10 ng/ml) inhibition of SP-LI release. Basal and electrically evoked release of SP-LI from the rat spinal cord in vitro was not modified 24 hr after single systemic injection of either NGF (1 mg/kg) or NT-3 (10 mg/kg). At these time intervals from administration, NGF had induced thermal and mechanical hyperalgesia in the rat hindpaw, and NT-3 had induced mechanical, but not thermal, hypoalgesia. NT-3 administered six times over a 2 week period (at 1 mg/kg) did not alter thermal threshold but significantly reduced electrically evoked release of SP-LI from the spinal cord. An identical treatment regimen with 1 mg/kg NGF induced a significant increase in evoked release of SP-LI. However, this was not associated with a significant hyperalgesia. Although finding that NGF-induced hyperalgesia does not clearly correlate with changes in the release of SP-LI in the spinal cord, this study shows that NT-3 is an inhibitor of SP-LI release and suggests that this mechanism may be responsible for NT-3-induced antinociception.

Effect of capsaicin on substance P and nerve growth factor in adjuvant arthritic rats.

We have investigated the effect of capsaicin pretreatment (50 mg kg(-1) s.c.) on substance P, preprotachykinin (PPT) mRNA, and nerve growth factor (NGF), plus its high-affinity receptor, trkA, in adult rats with adjuvant arthritis. Twenty one days after induction of adjuvant arthritis, sciatic nerve levels of substance P were significantly increased whilst there was a small but non-significant increase in gamma-PPT mRNA and substance P in L4/L5 dorsal root ganglia (DRG). NGF levels in sciatic nerve and foot skin as well as DRG trkA mRNA were unaltered after 21 days arthritis suggesting that NGF may not play a role in chronic inflammation. Capsaicin treatment of naive rats significantly reduced substance P in all tissues and NGF levels in the sciatic nerve. In contrast, gamma-PPT mRNA and trkA mRNA expression in DRG were significantly increased after capsaicin treatment. The nervous and skin tissues used in this study were harvested from the same rats in which we had previously shown that capsaicin pretreatment significantly attenuated the severity of arthritis (Cruwys, S.C., Garrett, N.E. and Kidd, B.L., Sensory denervation with capsaicin attenuates inflammation and nociception in arthritic rats, Neurosci. Lett., 193 (1995) 205-207). Arthritis in capsaicin-treated rats had no effect on substance P or NGF levels in any tissue when compared with capsaicin-treated control rats, suggesting that pharmacological impairment of the sensory nervous system can reduce the severity of inflammatory joint disease.

Nerve growth factor treatment increases stimulus-evoked release of sensory neuropeptides in the rat spinal cord.

In the adult rat, nerve growth factor (NGF) upregulates the nociceptive peptide substance P (SP) and calcitonin gene-related peptide (CGRP) in neuronal cell bodies of C fibres but the effects of NGF on release of these neuropeptides from the central afferent terminals have not been reported. Thus, this study compared rats treated with six doses of NGF over 2 weeks with controls and measured the release of the neuropeptides, SP and CGRP from the dorsum of the lumbar spinal cord in vitro. NGF (1.0 mg/kg s.c.) greatly increased basal and stimulus-evoked SP and CGRP release; at 0.1 mg/kg, NGF did not affect SP release but significantly increased CGRP basal outflow and evoked release. In a different set of experiments, topical application of NGF (100 ng/ml) to cords from naive rats did not increase stimulus-evoked release of SP or CGRP. These findings show marked stimulation by NGF treatment in vivo of release of sensory neuropeptides during stimulation of dorsal roots, albeit at relatively large doses of the neurotrophin.

alpha-Lipoic acid corrects neuropeptide deficits in diabetic rats via induction of trophic support.

This study compared the effects of treatment of diabetic rats with either alpha-lipoic acid (100 mg/kg/day i.p. 5 days/week) or with recombinant human nerve growth factor (rhNGF; 0.2 mg/kg s.c. 3 days/week) on NGF-like immunoreactivity (NGFLI) and neuropeptide Y-like immunoreactivity (NPYLI) levels in the sciatic nerve and on the release of substance P-like immunoreactivity (SPLI) from the spinal cord in response to electrical stimulation of the dorsal roots in vitro. Diabetic rats showed depletion of NGFLI and NPYLI, together with reduced release of SPLI. Treatment with NGF increased the sciatic nerve NGFLI (to four times that seen in untreated diabetic rats) and normalised stimulus-evoked release of SPLI, but did not affect the sciatic nerve NPYLI. Treatment with alpha-lipoic acid caused a small non-significant increase in sciatic nerve NGFLI, but normalised both NPYLI levels and stimulus-evoked release of SPLI. These findings indicate that alpha-lipoic acid can boost neurotrophic support in diabetic rats, with effects beyond those related to NGF.

Effect of streptozotocin-diabetes on knee joint inflammation-induced changes in substance P and nerve growth factor in the rat.

Given the involvement of the sensory nervous system in the aetiology of neurogenic inflammation, we have investigated the effect of experimental diabetes and any associated sensory nerve dysfunction on the development of complete Freund's adjuvant-induced inflammation in the rat knee. Twenty-four hours after induction of inflammation in non-diabetic rats, gamma-preprotachykinin mRNA expression was increased in the L4/L5 dorsal root ganglia. Substance P levels were increased in dorsal root ganglia and sciatic nerve whilst synovial levels of substance P were significantly decreased. Nerve growth factor, which regulates expression of gamma-preprotachykinin mRNA, was significantly increased in synovium and sciatic nerve after induction of inflammation. After 24 weeks of streptozotocin-diabetes, there was a non-significant reduction in gamma-preprotachykinin mRNA expression whilst substance P levels in dorsal root ganglia, sciatic nerve and synovium and nerve growth factor levels in the sciatic nerve were significantly decreased. Conversely, synovial levels of nerve growth factor were significantly increased. Injection of complete Freund's adjuvant into the knee of diabetic rats produced diminished joint swelling compared to that observed in non-diabetic rats. Substance P levels were unaltered compared to non-arthritic diabetic rats whilst nerve growth factor levels were significantly increased in synovium and sciatic nerve suggesting an uncoupling of substance P from nerve growth factor control in the inflammatory response in diabetic rats. The results show a significant reduction in the inflammatory response in rats with chronic streptozotocin-diabetes. Deficits in gamma-preprotachykinin mRNA expression and substance P and the altered levels of nerve growth factor indicate sensory neuronal dysfunction may play a major role in this abnormal response.

Neurogenic influences on contralateral responses during experimental rat monoarthritis.

Many inflammatory conditions show topographically precise symmetrical responses. In this study we assessed vascular and cellular responses of apparently normal knees following induction of monoarthritis on the opposite side. A strictly localised monoarthritis was induced in the right knee of experimental animals using intra-articular latex spheres. In both knee joints bradykinin-induced plasma extravasation was significantly enhanced increasing from 0.52 +/- 0.07 micrograms/ml Evans blue to 0.99 +/- 0.07 micrograms/ml and 0.88 +/- 0.1 micrograms/ml in the injected and uninjected, contralateral, knees respectively (P < 0.05). A bilateral increase in cellularity was also apparent with cell counts in the uninjected, and apparently normal, knee increasing from 512 +/- 42 cells/mm2 to a maximum of 812 +/- 125 cells/mm2 on day 10 (P < 0.05). Immunohistological analysis demonstrated that the infiltrating cells in both the ipsilateral and contralateral joints were predominantly macrophages. Cell counts were not increased in the other peripheral joints. Levels of the sensory neuropeptide substance P were significantly elevated in both the ipsilateral and contralateral dorsal root ganglia and prior inhibition of small unmyelinated nerve activity inhibited the cellular infiltrate on the contralateral side, suggesting that the effect was mediated, at least partially, by a specific neurogenic pathway. The data suggests the presence of a neurogenic mechanism able to induce a topographically precise response. This may serve to upregulate the cellular defences of at-risk tissues following a potentially damaging stimulus at another site.

Sensory denervation with capsaicin attenuates inflammation and nociception in arthritic rats.

The relatively few studies that have investigated the effects of the nervous system on chronic joint disease have reported conflicting results. We have reassessed the effects of capsaicin on experimental polyarthritis with particular reference to the relationship between changes in nociception and changes in process and outcome measures of disease activity. Capsaicin pretreatment significantly attenuated both joint swelling and disease outcome as determined by quantitative radiology and histology. There was a close correlation between process measures of inflammation and mechanical hyperalgesia in both the untreated and capsaicin treated arthritic groups. The results confirm a suppression of inflammation by capsaicin and imply that the nociceptive and pro-inflammatory (neurogenic inflammation) activities of capsaicin-sensitive fibres are closely linked such that stimuli which cause pain will also induce neurogenic inflammation and vice versa.

Streptozotocin-induced diabetes decreases substance P levels in experimental arthritis in the rat knee.

Diabetic patients with sensory neuropathy are predisposed to disorders of the musculoskeletal system. It has been postulated that altered neurogenic inflammation, involving the neuropeptide substance P, may play a part in this phenomenon. We investigated the effect of streptozotocin-induced (STZ) diabetes on the development of an antigenic (mBSA) monoarthritis in the rat with particular reference to changes in substance P levels in dorsal root ganglia (DRG) and knee joint synovium. We found that STZ-induced diabetes of 24 weeks duration reduced the substance P content of L4/L5 DRG and knee joint synovial tissue. Induction of mBSA arthritis in diabetic rats resulted in diminished increases in synovial substance P and knee joint swelling compared to non-diabetic arthritic controls. The results show that chronic STZ diabetes reduces neurogenic inflammatory responses in the rat knee which may render the joint more susceptible to arthritic attack.

Changes in preprotachykinin mRNA expression and substance P levels in dorsal root ganglia of monoarthritic rats: comparison with changes in synovial substance P levels.

We have measured changes in the expression of gamma-preprotachykinin mRNA and levels of the neuropeptide substance P in the lumbar 4 and 5 dorsal root ganglia at various time points following the induction of an antigenic monoarthritis in the rat knee. The results were compared with changes in substances P levels in the knee joint synovium during acute and chronic phases of the disease. On day 3 post-induction, there was a significant increase in the expression of gamma-preprotachykinin mRNA in the dorsal root ganglia. Concomitant with this increase in message was a rise in the levels of substance P in the dorsal root ganglia. On days 7, 10 and 21, mRNA expression had returned to control values whereas ganglion peptide levels were significantly below controls. In contrast there was little change in the total substance P levels in the synovium on days 1 and 3 despite the observed changes in the ganglia. By day 10, however, synovial levels had risen significantly above control values and remained elevated thereafter. Our results show a transitory increase in substance P synthesis after induction of an antigenic monoarthritis. This response is not mirrored in the periphery where there is no initial change in total substance P levels perhaps reflecting increased degradation be enzymes known to be present within inflamed tissue. Paradoxically synovial substance P levels are increased in the latter phases of the model which may serve to modify the inflammatory response.

The role of bradykinin B1 receptors in the maintenance of intra-articular plasma extravasation in chronic antigen-induced arthritis.

1. The role of bradykinin B1 and B2 receptors in bradykinin- and des-Arg9-bradykinin-induced plasma extravasation in normal and inflamed rat knee joints was investigated by use of an antigen-induced model of chronic arthritis. A modification of an Evans blue extraction technique allowed the unstimulated (basal) plasma extravasation to be assessed in this model. The contributions of bradykinin B1 and B2 receptors towards basal synovial plasma extravasation were determined. 2. In normal knees, intra-articular injection of bradykinin (BK) induced plasma extravasation in a potent, dose-dependent manner with a threshold of 0.01 nmol and an ED50 of 0.1 nmol. In day 5 arthritic knees, basal plasma extravasation was substantially enhanced. Lower doses of BK had no demonstrable effect and increases above basal extravasation were first observed at 0.1 nmol. Thereafter the dose-response mirrored the response in normal knees and the maximal response was unaltered. 3. The B1 agonist, des-Arg9-BK, induced slight but significant plasma extravasation in normal knees but was less potent than bradykinin. This response was inhibited by the B1 receptor antagonist, des-Arg9, [Leu8]-BK. Lower doses of des-Arg9-BK bradykinin did not significantly increase basal extravasation in day 5 arthritic knees but, in contrast to BK, the maximal response was significantly enhanced. 4. The B2 antagonist, Hoe 140, inhibited BK-induced plasma extravasation in normal joints over a dose-range of 0.1-1.0 nmol but was relatively inactive in day 5 inflamed knees. The B1 receptor antagonist, des-Arg9, [Leu8]-BK, was relatively inactive in normal joints but showed increased potency against BK-induced plasma extravasation in day 5 arthritic joints.5. Hoe 140 and des-Arg9,[Leu8]-BK both inhibited basal extravasation in arthritic joints on days 1 and 5 post-challenge in a dose-dependent fashion. Whilst Hoe 140 was the more potent inhibitor on day 1, it was less potent than des-Arg9,[Leu8]-BK on day 5.6. Although the majority of responses to BK in normal tissue are mediated via B2 receptors, a small population of B1 receptors may exist in normal joint tissues. The data presented in this study suggest an evolving role for B1 receptors in the mediation of plasma extravasation in inflamed joint tissues. A role for BK antagonists in the treatment of arthritis is also suggested.

Effect of three animal models of inflammation on nerve fibres in the synovium.

OBJECTIVES: Both sensory and sympathetic nerve fibres are depleted in the synovium in rheumatoid arthritis (RA). The hypothesis that the induction of an inflammatory response in the synovium is capable of causing depletion of nerve fibres was tested. METHODS: To investigate this phenomenon experimental arthritis in the rat was induced by three different methods and the synovium was examined for evidence of nerve depletion by immunocytochemistry. RESULTS: In a synovitis induced by latex spheres, a mainly macrophage foreign body type reaction, no nerve depletion was seen. In contrast both in an antigen-induced and a hydrogen peroxide-induced model of arthritis nerve fibre depletion was observed. This appeared to affect sensory and sympathetic nerve fibres equally. Nerve fibre depletion was only seen in areas of inflammatory cell infiltration indicating that a mixed lymphocyte and macrophage population of cells may be necessary for this effect. CONCLUSIONS: An inflammatory response, containing lymphocytes and macrophages, in the synovium is capable of the depletion of the finely myelinated and unmyelinated neuropeptide-containing nerves.

The Genetic Activity Profile database.

A graphic approach termed a Genetic Activity Profile (GAP) has been developed to display a matrix of data on the genetic and related effects of selected chemical agents. The profiles provide a visual overview of the quantitative (doses) and qualitative (test results) data for each chemical. Either the lowest effective dose (LED) or highest ineffective dose (HID) is recorded for each agent and bioassay. Up to 200 different test systems are represented across the GAP. Bioassay systems are organized according to the phylogeny of the test organisms and the end points of genetic activity. The methodology for the production and evaluation of GAPs has been developed in collaboration with the International Agency for Research on Cancer. Data on individual chemicals have been compiled by IARC and by the U.S. Environmental Protection Agency. Data are available on 299 compounds selected from volumes 1-50 of the IARC Monographs and on 115 compounds identified as Superfund Priority Substances. Software to display the GAPs on an IBM-compatible personal computer is available from the authors. Structurally similar compounds frequently display qualitatively and quantitatively similar GAPs. By examining the patterns of GAPs of pairs and groups of chemicals, it is possible to make more informed decisions regarding the selection of test batteries to be used in evaluating chemical analogs. GAPs have provided useful data for the development of weight-of-evidence hazard ranking schemes. Also, some knowledge of the potential genetic activity of complex environmental mixtures may be gained from assessing the GAPs of component chemicals. The fundamental techniques and computer programs devised for the GAP database may be used to develop similar databases in other disciplines.

Genetic activity profiles and pattern recognition in test battery selection.

Computer-generated genetic activity profiles and pairwise matching procedures may aid in the selection of the most appropriate short-term bioassays to be used in test batteries for the evaluation of the genotoxicity of a given chemical or group of chemicals. Selection of test batteries would be based on a quantitative comparative assessment of the past performance of similar tests applied to other chemicals of the same structural group. The information potentially available for test-battery selection through the use of this pattern-recognition technique is considerably greater than the qualitative results obtained from individual short-term tests. Application of the method should further our understanding of the relationships between chemical properties and genotoxic responses obtained in short-term bioassays and also may contribute to our knowledge of the mechanisms of complex processes such as carcinogenesis. This approach to battery selection should be augmented by careful consideration of established principles of genetic toxicity testing; that is, a chemical should be evaluated in a battery of tests representing the full range of relevant genetic endpoints.

Evaluation of the genetic activity profiles of 65 pesticides.

We have previously reported the qualitative results of a major study on 65 pesticides (Waters et al., 1982). Dose information from this investigation (either lowest effective or highest ineffective dose tested) has now been incorporated into a computerized data management system. This report focuses on the qualitative profiles of genetic activity produced by these pesticides and our efforts to classify them according to their genotoxic effects and chemical structures. Three main categories may be distinguished based on the qualitative results: Category 1 pesticides were active in most of the in vitro and in vivo assays employed. These 9 compounds include the structurally similar organophosphate insecticides, acephate, demeton, monocrotophos and trichlorfon; the phthalimide fungicide analogues, captan and folpet; and the thiocarbamate herbicide analogues, diallate, sulfallate and triallate. The 26 Category 2 compounds demonstrated fewer positive results and may be subdivided into two parts, one of which contains 12 halogenated aromatic or heterocyclic ring compounds, including the phenoxy herbicides, 2,4-D, 2,4-DB and 2,4,5-T. The remaining part of Category 2 (14 compounds) consists of structurally similar organophosphate insecticides, azinphos-methyl, crotoxyphos, disulfoton, methyl parathion; three similar ethylenebisdithiocarbamate fungicides, maneb, mancozeb, and zineb; three similar pyrethroid insecticides, allethrin, chrysanthemic acid, and ethyl chrysanthemate; and four structurally diverse compounds, cacodylic acid, dinoseb, sec.-butylamine and benomyl. The third category of 30 pesticides gave negative results in all tests and represents structurally diverse compounds. Using the computerized profile matching methodology, from 2080 possible pairwise chemical combinations of the 65 pesticides, 20 statistically significant pairs were selected, 6 groups of pesticides were identified which were substantially similar to groups of pesticides we had formed previously (Waters et al., 1982) based on genetic activity and chemical structure. The matches showed excellent qualitative and, in most cases, excellent quantitative agreement. Hence it appears that specific patterns of test results present in the genetic activity profiles are related directly to chemical structure. Conversely, the data suggests that certain groups of compounds may be recognized by a well defined series of concordant tests results. As additional data is added, comparison of test results for new chemicals with existing data for known genotoxicants should aid in the evaluation of potential genetic health hazards.

An analysis of the spectra of genetic activity produced by known or suspected human carcinogens.

For 24 agents classified by the International Agency for Research on Cancer as known or suspected human carcinogens, we previously catalogued the qualitative genetic bioassay data available in the literature. In the present analysis, dose information, where available, was added to this data base: either the lowest effective dose (LED) or the highest ineffective dose (HID) was recorded for each agent and bioassay system. Bioassay systems were organized according to classes of genetic activity and subdivided by the phylogenetic level of the test organism. For each compound, the quantitative results in the test systems were represented by computer-generated bar graphs ('genetic activity spectra'). The x-axis unit values corresponded to the 100 different test systems, and the y-axis values were the logarithmically transformed LED or HID values. Statistical methods and pattern-recognition techniques were used to evaluate the genetic activity spectra. Spectra were compared among agents grouped according to target-organ specificity. In addition, the spectra of all possible pairs of compounds were compared to identify compounds displaying qualitatively or quantitatively similar genetic activity. Chemically similar compounds frequently produced similar spectra of genetic activity, and it was possible to identify the most appropriate test systems for some classes of compounds. As the data base for human carcinogens is enlarged, analysis of genetic activity spectra may contribute to our understanding of the structure-activity relationships and mechanisms of action of these agents.

Using the desk top computer in cellular toxicity and mutagenesis.

A series of programs applicable to in vitro test symptoms in environmental toxicology are described for the Tektronix 4050 series graphic system computer. The file structure, experimental design and identification, the program library, data entry program, and programs to compile data from separate experiments are presented. Experimental design information and test data are stored on magnetic tape. The programs are designed to compute cell number and viability, adenosine triphosphate level, and protein and DNA synthetic activity from raw data obtained from cellular toxicity experiments; and cloning efficiency, mutation yield, mutation frequency, and other parameters for mutagenicity. A set of subprograms can be used for statistical analysis of the data and for construction of frequency distributions. The applicability of the programs is illustrated by data obtained from exposure of Chinese hamster ovary cells to cadmium chloride or N-methyl-N'-nitro-N-nitroso-guanidine.

Cellular toxicity in Chinese hamster ovary cell cultures. I. Analysis of cytotoxicity endpoints for twenty-nine priority pollutants.

Chinese hamster ovary cells were exposed to 29 toxic chemical substances which were representative of several classes of compounds listed by the Natural Resources Defense Council Consent Decree as priority toxic pollutants. After cell cultures were exposed to the test substance, cell samples were assayed for protein and DNA synthesis, ATP, cell number, and viability. A filter-disk technique employing a batch-washing procedure was used for the determination of protein and DNA synthesis. Dose-response data were obtained for 15 of the more toxic agents including chlorinated aromatics, metallic compounds, phenols, and polychlorinated biphenyls. Estimates of the sample concentrations necessary to produce a 50% reduction in response were used to compare cytotoxicity endpoints. ATP and protein synthesis were approximately equally effective as indicators of cellular toxicity. Cadmium chloride, nickel nitrate, arsenic trioxide, and potassium chromate produced a more pronounced effect on DNA synthesis than on ATP or protein synthesis. The dose-response relationship for protein and DNA synthesis was a smooth, continuous function for responses as low as 1 to 2% of the control. On a log-log scale, the dose-response relation yielded a unique pattern for several chemical classes. A ranking of the compounds based on their inhibition of DNA synthesis was compared to a ranking obtained from the literature for whole-animal toxicity. With the exception of the polychlorinated biphenyls, the in vitro results correlated well with animal test data.

Cellular toxicity in Chinese hamster ovary cell cultures. II. A statistical appraisal of sensitivity with the rabbit alveolar macrophage, Syrian hamster embryo, BALB 3T3 mouse, and human neonatal fibroblast cell systems.

Chinese hamster ovary, rabbit alveolar macrophage, Syrian hamster embryo, BALB 3T3 mouse, and human neonatal fibroblast cells were employed in a statistical evaluation of the relative sensitivity of the cells to toxic substances. The cells were exposed to 1,2,4-trichlorobenzene, 2,4-dimethylphenol, Aroclor 1248, cadmium chloride, lead sulfate, nickel nitrate, lead oxide-coated fly ash, and a fine particulate from coal combustion. A filter-disk technique was used to measure the inhibition of protein and DNA synthesis. A quantitative ranking of cell-system sensitivity was determined from comparisons of statistically significant differences (P less than or equal to 0.01) in protein and DNA synthesis expressed as a percentage of control. An overall ranking of sensitivity showed that rabbit alveolar macrophages, Syrian hamster embryo cells, and Chinese hamster ovary cells were more sensitive than another of the five cell systems in 75, 68, and 62% of the experiments, respectively. The corresponding values for BALB 3T3 mouse and human neonatal fibroblast cells were 38 and 28%, respectively, under our experimental conditions. Detailed data on the control cell cultures are also presented.