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There is an ongoing debate about the 'usefulness' of the standard machine smoking regimen for cigarettes defined by the US Federal Trade Commission (FTC) and adopted in principal by the International Organization for Standardization (ISO). More intense smoking regimens result in much higher smoke yields, and these higher yields have been suggested to be much closer to human smoke uptake. However, it appears that more intense smoking regimens are less efficient in detecting possible differences in the yield of toxicants. Intense smoking regimens reproducibly decrease the concentration of toxicants in the smoke per unit mass of total particulate matter, tar, or nicotine, most likely as a result of a more complete combustion. The toxicant concentration reaches the same plateau for different cigarette types under the intense smoking regimens. As such, differences in toxicant concentrations due to product changes, which are observable under ISO or FTC conditions, may disappear under intensive smoking regimens. Intense machine smoking regimens might be used for regulatory compliance and consumer information. However, when evaluating the toxicological impact of cigarette product changes, especially to avoid increases in toxicity, they should not be used as a standard (or at least not as the only standard). The type of smoking regimen has to be carefully considered and aligned to the underlying question. Depending on the question a combination of the reviewed regimens or even other approaches might be appropriate., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

In a previous analysis (see Part I) we proposed a heuristic for assessing the efficacy of potential reduced-risk tobacco products (PRRPs) on lung cancer (LC) rates, using smoking cessation data published in a report from the Iowa Women's Health Study (IWHS) as a basis for sample size estimates. In this study, an additional analysis was performed using cessation data from the much larger Cancer Prevention Study II (CPS-II), which also provides data on different durations of cessation. Statistical methods were used to assess whether smokers switching to a PRRP would reduce their risk of LC. Furthermore, non-inferiority tests compared the LC risk in switchers to that in smokers who had quit smoking. The present work shows that similar sample size estimates were obtained whether the analysis was based on the IWHS or the CPS-II data sets, suggesting that the heuristic may be generally applicable to prospective real-life studies to evaluate PRRPs. Non-inferiority testing of switchers compared with quitters required approximately 10-fold more subjects than did superiority testing of switchers compared with smokers. Altogether, these estimates indicate that it is feasible, in terms of study duration and sample size, to clinically assess the LC risk-reducing potential of a PRRP., ((c) 2009 Elsevier Inc. All rights reserved.)

The risk-reducing effect of a potential reduced-risk tobacco product (PRRP) can be investigated conceptually in a long-term, prospective study of disease risks among cigarette smokers who switch to a PRRP and in appropriate comparison groups. Our objective was to provide guidance for establishing the fundamental design characteristics of a study intended to (1) determine if switching to a PRRP reduces the risk of lung cancer (LC) compared with continued cigarette smoking, and (2) compare, using a non-inferiority approach, the reduction in LC risk among smokers who switched to a PRRP to the reduction in risk among smokers who quit smoking entirely. Using standard statistical methods applied to published data on LC incidence after smoking cessation, we show that the sample size and duration required for a study designed to evaluate the potential for LC risk reduction for an already marketed PRRP, compared with continued smoking, varies depending on the LC risk-reducing effectiveness of the PRRP, from a 5-year study with 8000-30,000 subjects to a 15-year study with <5000 to 10,000 subjects. To assess non-inferiority to quitting, the required sample size tends to be about 10 times greater, again depending on the effectiveness of the PRRP., ((c) 2009 Elsevier Inc. All rights reserved.)

Rationale: Growing proportions of smokers in the USA do not smoke everyday and can be referred to as light and intermittent smokers (LITS). Despite a current prevalence of LITS in the USA estimated at 25-33% of all smokers, a systematic review of the literature on this group of smokers has yet to be written., Objectives: The aim of this paper is to review and evaluate research on LITS and to identify, describe and discuss commonalities and differences between LITS and daily smokers., Methods: The primary databases used to search for publications were Pub Med (National Library of Medicine) and SCOPUS (Elsevier)., Results: LITS inhale smoke and have post-smoking blood nicotine concentrations that are broadly equivalent to those found in daily smokers. However, LITS differ from daily smokers with regard to cigarette consumption and frequency of cigarette use, sociodemographic and socioeconomic characteristics, motives, personality traits, dependence, withdrawal and craving, response to smoking-related cues, quitting perception, past-smoking status, and initiation., Conclusions: In contrast to daily smokers, LITS show few or no signs of dependence as currently defined by DSM-IV criteria, appear to exercise more self-control, seem to be less impulsive, and their smoking experience is primarily associated with positive rather than negative reinforcement. Conclusions drawn from the reviewed literature highlight the multivariate factors that must be taken into account when defining LITS and emphasize the importance of further research on this increasing fraction of smokers. The potential implications of increased LITS prevalence on smoking-related disease risks remain to be thoroughly investigated.

The particle phase of mainstream smoke from three types of cigarettes was investigated in vitro in the Neutral Red cytotoxicity assay and the Salmonella typhimurium Reverse Mutation Assay (Ames Assay) and in vivo in the two-stage dermal tumorigenicity assay (Skin Painting Assay) in SENCAR mice. The cigarettes used were the Reference Cigarettes 1R5F, 2R4F, and 2R1F from the University of Kentucky, USA, which, when smoked according to the smoking regimen defined by the International Standards Organization (ISO), produce a yield of approximately 2, 12, and 26 mg total particulate matter (TPM)/cigarette, respectively. All cigarettes were machine smoked according to ISO and then again in such a way that the TPM yields per cigarette equaled the ISO TPM yields of the other two cigarette types. The TPM from cigarettes with inherently different smoke yields showed similar in vitro toxicity and in vivo toxicity when, with different smoking regimens, these cigarettes were smoked to the same TPM yield. More intensive smoking conditions were associated with lower in vitro and in vivo activity per gram of TPM. The strongest decrease, and the tightest correlation, in this regard was observed for dermal tumorigenicity (tumor incidence).

We compared risk of lung cancer and chronic obstructive pulmonary disease (COPD) associated with flue-cured and blended cigarettes. Mortality and smoking data were collected for 1971-2000 by sex, age, and period for three countries with a mainly flue-cured market and four with a blended market. Epidemiological relative risk estimates for current and ex smoking were summarized. Smoking statistics and mortality were compared between flue-cured cigarette and blended cigarette countries. Unadjusted mortality rates were generally lower in blended cigarette countries early on, with the difference diminishing or reversing by the 1990s. Differences by cigarette type were rarely significant, due to variations, particularly for COPD, between countries within cigarette type. Current smoking prevalence was generally lower in blended cigarette countries in 1971-1975, with the difference reducing over time. Differences by type were never significant, with blended cigarette countries varying markedly. Ex-smoking increased over time and was lower for blended cigarette countries, generally not significantly. Consumption per smoker was somewhat lower for blended cigarette countries. Relative risk estimates for smoking, derived mainly from U.S. and UK studies, varied little by cigarette type. Conclusions based on estimated smoking-related excess mortality were similar to those based on unadjusted mortality rates. There was little indication of any difference between flue-cured and blended cigarettes on risk of lung cancer or COPD. Our approach could have detected differences of about 40% for male lung cancer, or twofold differences for females or for COPD, had they existed. Between-country differences in rates of two major diseases predominantly caused by smoking cannot materially be explained by whether the countries use flue-cured or blended cigarettes.

A complete and rigorous review is presented of the possible effect(s) of ammonia on the exposure, deposition and retention of nicotine during smoking and the bioavailability of nicotine to the smoker. There are no toxicological data in humans regarding ammonia exposure within the context of tobacco smoke. Extrapolation from occupational exposure of ammonia to smoking in humans suggests minimal, non-toxicological effects, if any. No direct study has examined the effect of the ammonia on the total rate or amount of nicotine reaching the arterial bloodstream or brains of smokers. Machine-smoking methods have been reported which accurately quantify >99% of the nicotine in mainstream (MS) smoke for a wide variety of commercial and test cigarettes, including a series of experimental cigarettes having a range in MS smoke ammonia yields using the US Federal Trade Commission (FTC) protocol. However, the actual exposure of nicotine to smokers depends on their own smoking behavior. The nicotine ring system is relatively thermally stable. Protonated nicotine forms nicotine which evaporates before the nicotine ring system decomposes. The experimental data indicate that neither nicotine transfer from tobacco to MS smoke nor nicotine bioavailability to the smoker increases with an increase in any of the following properties: tobacco soluble ammonia, MS smoke ammonia, "tobacco pH" or "smoke pH" at levels found in commercial cigarettes. Gas phase nicotine deposits primarily in the mouth and upper respiratory tract. To the extent that ammonia increases the deposition of nicotine in the buccal cavity and upper respiratory tract during smoking, the total rate and amount of nicotine into the arterial bloodstream and to the central nervous system will decrease. Charged nicotine analogues are actively transported in a number of tissues. This active transport system appears to be insensitive to pH and the form of nicotine in the biological milieu, suggesting that protonated nicotine may be a substrate for active transport. Neither "smoke pH" of commercial cigarettes nor "smoke pHeff" nor the fraction of non-protonated nicotine in tobacco smoke particulate matter are useful, practical smoke parameters for providing understanding or predictability of nicotine bioavailability to smokers. Greater than 95% of both ammonia and nicotine are in the gas phase of environmental tobacco, and both are likely to deposit in the buccal cavity and upper respiratory tract following exposure.

Several assumptions, defined and undefined, are used in the toxicity assessment of chemical mixtures. In scientific practice mixture components in the low-dose region, particularly subthreshold doses, are often assumed to behave additively (i.e., zero interaction) based on heuristic arguments. This assumption has important implications in the practice of risk assessment, but has not been experimentally tested. We have developed methodology to test for additivity in the sense of Berenbaum (Advances in Cancer Research, 1981), based on the statistical equivalence testing literature where the null hypothesis of interaction is rejected for the alternative hypothesis of additivity when data support the claim. The implication of this approach is that conclusions of additivity are made with a false positive rate controlled by the experimenter. The claim of additivity is based on prespecified additivity margins, which are chosen using expert biological judgment such that small deviations from additivity, which are not considered to be biologically important, are not statistically significant. This approach is in contrast to the usual hypothesis-testing framework that assumes additivity in the null hypothesis and rejects when there is significant evidence of interaction. In this scenario, failure to reject may be due to lack of statistical power making the claim of additivity problematic. The proposed method is illustrated in a mixture of five organophosphorus pesticides that were experimentally evaluated alone and at relevant mixing ratios. Motor activity was assessed in adult male rats following acute exposure. Four low-dose mixture groups were evaluated. Evidence of additivity is found in three of the four low-dose mixture groups. The proposed method tests for additivity of the whole mixture and does not take into account subset interactions (e.g., synergistic, antagonistic) that may have occurred and cancelled each other out.

Robust statistical methods are important to the evaluation of toxicological interactions (i.e., departures from additivity) among chemicals in a mixture. However, different concepts of joint toxic action as applied to the statistical analysis of chemical mixture toxicology data or as used in environmental risk assessment often appear to conflict with one another. A unifying approach for application of statistical methodology in chemical mixture toxicology research is based on consideration of change(s) in slope. If the slope of the dose-response curve of one chemical does not change in the presence of other chemicals, then there is no interaction between the first chemical and the others. Conversely, if the rate of change in the response with respect to dose of the first chemical changes in the presence of the other chemicals, then an interaction is said to exist. This concept of zero interaction is equivalent to the usual approach taken in additivity models in the statistical literature. In these additivity models, the rate of change in the response as a function of the i(th) chemical does not change in the presence of other chemicals in a mixture. It is important to note that Berenbaum's (1985, J. Theor. Biol. 114, 413-431) general and fundamental definition of additivity does not require the chemicals in the mixture to have a common toxic mode of action nor to have similarly shaped dose response curves. We show an algebraic equivalence between these statistical additivity models and the definition of additivity given by Berenbaum.

Endocrine disruption from environmental contaminants has been linked to a broad spectrum of adverse outcomes. One concern about endocrine-disrupting xenobiotics is the potential for additive or synergistic (i.e., greater-than-additive) effects of mixtures. A short-term dosing model to examine the effects of environmental mixtures on thyroid homeostasis has been developed. Prototypic thyroid-disrupting chemicals (TDCs) such as dioxins, polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers have been shown to alter thyroid hormone homeostasis in this model primarily by up-regulating hepatic catabolism of thyroid hormones via at least two mechanisms. Our present effort tested the hypothesis that a mixture of TDCs will affect serum total thyroxine (T4) concentrations in a dose-additive manner. Young female Long-Evans rats were dosed via gavage with 18 different polyhalogenated aromatic hydrocarbons [2 dioxins, 4 dibenzofurans, and 12 PCBs, including dioxin-like and non-dioxin-like PCBs] for 4 consecutive days. Serum total T4 was measured via radioimmunoassay in samples collected 24 hr after the last dose. Extensive dose-response functions (based on seven to nine doses per chemical) were determined for individual chemicals. A mixture was custom synthesized with the ratio of chemicals based on environmental concentrations. Serial dilutions of this mixture ranged from approximately background levels to 100-fold greater than background human daily intakes. Six serial dilutions of the mixture were tested in the same 4-day assay. Doses of individual chemicals that were associated with a 30% TH decrease from control (ED30), as well as predicted mixture outcomes were calculated using a flexible single-chemical-required method applicable to chemicals with differing dose thresholds and maximum-effect asymptotes. The single-chemical data were modeled without and with the mixture data to determine, respectively, the expected mixture response (the additivity model) and the experimentally observed mixture response (the empirical model). A likelihood-ratio test revealed statistically significant departure from dose additivity. There was no deviation from additivity at the lowest doses of the mixture, but there was a greater-than-additive effect at the three highest mixtures doses. At high doses the additivity model underpredicted the empirical effects by 2- to 3-fold. These are the first results to suggest dose-dependent additivity and synergism in TDCs that may act via different mechanisms in a complex mixture. The results imply that cumulative risk approaches be considered when assessing the risk of exposure to chemical mixtures that contain TDCs.

Nose-only exposure of male and female Wistar rats to a surrogate for environmental tobacco smoke, termed room-aged sidestream smoke (RASS), to diesel engine exhaust (DEE), or to filtered, fresh air (sham) was performed 6 hours/day, 7 days/week for 2 years, followed by a 6-month post-exposure period. The particulate concentrations were 3 and 10 mg/m3. Markers of inflammation in bronchoalveolar lavage showed that DEE (but not RASS) produced a dose-related and persistent inflammatory response. Lung weights were increased markedly in the DEE (but not RASS) groups and did not decrease during the 6-month post-exposure period. Bulky lung DNA adducts increased in the RASS groups, but not in the DEE groups. Cell proliferation in the lungs was unaffected by either experimental treatment. Histopathological responses in the RASS groups were minimal and almost completely reversible; lung tumors were similar in number to those seen in the sham-exposed groups. Rats exposed to DEE showed a panoply of dose-related histopathological responses: largely irreversible and in some cases progressive. Malignant and multiple tumors were seen only in the DEE groups; after 30 months, the tumor incidence (predominantly bronchiolo-alveolar adenomas) was 2% in the sham-exposed groups, 5%in the high RASS groups, and 46% in the high DEE groups (sexes combined). Our results suggest that in rats exposed to DEE, but not to RASS, the following series of events occurs: particle deposition in lungs --> lung "overload" --> pulmonary inflammation --> tumorigenesis, without a significant modifying role of cell proliferation or DNA adduct formation.

Assessing for interactions among chemicals in a mixture involves the comparison of actual mixture responses to those predicted under the assumption of zero interaction (additivity), based on individual chemical dose-response data. However, current statistical methods do not adequately account for differences in the shapes of the dose-response curves of the individual mixture components, as occurs with mixtures of full and partial receptor agonists. We present here a novel extension of current methods, which overcomes some of these limitations. Flexible single chemical concentration-effect curves combined with a common background parameter are used to describe an additivity surface along each axis. The predicted mixture response under the assumption of additivity is based on the constraint of Berenbaum's definition of additivity. Iterative algorithms are used to estimate mean responses at observed mixture combinations using only single chemical parameters. A full model allowing for different maximum response levels, different thresholds, and different slope parameters for each mixture component is compared to a reduced model under the assumption of additivity. A likelihood-ratio test is used to test the hypothesis of additivity by utilizing the full and reduced model predictions. This approach is useful for mixtures of chemicals with threshold regions and whose component chemicals exhibit differing response maxima (e.g., mixtures of full and partial agonists). The methods are illustrated with a combination of six chemicals in an estrogen receptor-alpha (ER-alpha) reporter gene assay.

Eight blended US market cigarettes, two blended reference cigarettes, one Bright tobacco only reference cigarette and an electrically heated prototype cigarette (EHC) were smoked under US Federal Trade Commission (FTC)/International Organisation for Standardisation (ISO) conditions and under Massachusetts Department of Public Health (MDPH) conditions. Smoke was analysed for chemical composition and in vitro toxicity. Yields (quantity/cigarette) of smoke constituents were higher under MDPH conditions compared to FTC/ISO conditions (market and reference average approximately 2.5 times; EHC approximately 1.6 times). Consistent with the higher yields, in vitro toxicity per cigarette was also higher under MDPH conditions. Concentrations (quantity/mg TPM) of nearly all smoke constituents measured decreased with increasing total particulate matter (TPM) yields as regression analyses indicated. Higher TPM yields also tended to be associated with slightly less cytotoxic and mutagenic activity per milligram TPM. Blended reference cigarettes tracked market cigarettes with similar TPM yield. The Bright cigarette displayed high cytotoxicity but low mutagenicity, while in vitro activity of the EHC was remarkably low. The TPM-dependent decreases for the market range of 5-20 mg TPM/cigarette were about 20%, irrespective of whether the increased yields were due to smoking conditions or cigarette construction. At the same TPM yield, the smoke constituent concentrations and in vitro toxicity were similar for low- and high-yield cigarettes.

Various combinations of carbamazepine (CAS 298-46-4), felbamate (CAS 25451-15-4), and phenytoin (CAS 57-41-0) were evaluated in mice (i.p.) for anticonvulsant activity (maximal electroshock seizure test) and minimal neurotoxicity (rotarod test). The results obtained from these studies were analyzed using response surface methodologies (RSM). The outcomes of these analyses in regard to anticonvulsant activity suggest that, under these experimental study conditions, at 0.5 h post treatment there is a significant carbamazepine/phenytoin synergism even though none of the drugs has a significant dose-response by that time when given alone, and that at 1.0 h post treatment, the combination dose-response is additive. Thus, there appears to be an important dose/time relationship. In regard to the neurotoxic response, the results suggest a significant carbamazepine/phenytoin synergism at 0.25 h post treatment and an additive neurotoxic effect due to the combination of felbamate/carbamazepine/phenytoin at 0.5, 1.0 and 2.0 h post exposure.

Angiofollicular lymph node hyperplasia is a heterogeneous disorder of unclear etiology and has a wide spectrum of systemic symptoms. This report describes a case of this disorder in a 15-year-old girl and examines the response of the primary mass, systemic symptoms, and alterations of selected immune parameters at diagnosis, as a result of steroid therapy and radiation therapy (RT). The patient had a 1-year history of growth failure, delayed puberty, and refractory iron deficiency anemia. Computed tomography scan showed a posterior mediastinal mass. Biopsy revealed angiofollicular lymph node hyperplasia of mixed hyaline-vascular and plasma cell type histologic type. Immunoperoxidase studies showed polyclonal B-cells, predominance of T-helper cells (CD4) over cytotoxic/suppressor T-cells (CD8), and the presence of natural killer (NK) cells. Southern blot analysis demonstrated germ line gene configuration for the T-cell antigen receptor and Ig heavy chain. The patient clinically improved with RT after failing to respond to steroids. Immunophenotyping of peripheral blood lymphocytes before therapy revealed a CD4:CD8 ratio of 0.8 with decreased numbers of circulating T-cells; this increased to 1.4 after steroid therapy. The patient's T-lymphocytes had no proliferative response to phytohemagglutinin (PHA) or concanavalin A (Con A) before RT. After RT, a small but significant mitogenic response to these reagents was noticed. The proliferative response to recombinant interleukin-2 (rIL-2) remained similar to that of control lymphocytes. Induction of second messenger signals by activation of protein kinase C (PKC) and elevation of free cytosolic calcium through the use of the phorbol ester, phorbol 12, 13-dibutyrate (PDBu), and ionomycin (Io) resulted in a strong proliferative response at diagnosis and after RT. In vitro cytotoxicity assays revealed diminished NK activity before and after therapy. Lymphokine-activated killer (LAK) activity remained comparable with that of control cells and was not affected by therapy. Before RT patient lymphocytes maintained cytotoxic capabilities after coincubation with rIL-2 and PDBu plus Io, whereas coincubation with these reagents abrogated cytotoxic function of normal cells. This case demonstrates a clinical response to RT as well as improvement in immune parameters. Intact signal transduction mechanisms through PKC activation and elevation of cytosolic calcium were also demonstrated in the circulating lymphocytes.

Menadione is a synthetic derivative of the natural vitamins K with antiinflammatory activity among its potentially significant clinical properties. We have found this agent to stimulate the production of superoxide anion (O2-) in human polymorphonuclear leukocytes (PMN) and dimethylsulfoxide-differentiated HL-60 cells in a time-, cell number-, and drug concentration-dependent manner. Conversely, menadione attenuates both O2- production and lysozyme release in cells stimulated by phorbol myristate acetate (PMA), fMet-Leu-Phe, or Ca2+ ionophore. 4-Acetamido-4'-isothiocyano-2-2'-disulfonic acid stilbene and 4,4'-diisothiocyano-2-2'disulfonic acid stilbene, agents which inhibit transmembrane O2-) flux, do not alter menadione's effects on superoxide dismutase (SOD) inhibitable cytochrome c reduction in resting or PMA-stimulated PMN. Likewise, quinone reductase inhibitors, warfarin and dicumarol, known to attenuate vitamin K-dependent responses and enhance quinone-mediated oxidative stress, have no effect upon menadione-stimulated O2- production. Furthermore, menadione-induced suppression of stimulus-mediated lysozyme release is not reversed by cotreatment with oxygen metabolite scavenging enzymes SOD and catalase. Nevertheless, under conditions of restricted oxygen supply, the suppressive effect of menadione on stimulant-induced lysozyme release is greatly diminished. Thus, although pharmacological manipulation suggests otherwise, there appears to exist at least a component of the inhibitory activity of menadione that is oxygen dependent, and may be oxidative stress-related.

Recent studies have shown that pretreatment with either pyridostigmine (PYR) or physostigmine (PHY) followed by atropine-oxime therapy is very effective in reducing the lethality of nerve agents. The therapeutic efficacy of a PHY and PYR combination pretreatment was evaluated in guinea pigs challenged with two LD50s of soman. Endpoints measured were percentage of acetylcholinesterase inhibition induced by the pretreatment and survival up to 24 hr postchallenge. Response surface methodology was employed to describe the relationship between each endpoint and the pretreatment combination. Although both carbamates contributed to blood acetylcholinesterase inhibition, PHY alone protected as well as the optimal dose of the combination.

This study was undertaken to determine whether the phorbol diester, phorbol 12-myristate 13-acetate (PMA), causes differentiation of the human colon carcinoma cell line, SW 48. Under routine growth conditions, the cells are round, have a high nuclear-to-cytoplasmic ratio, and lack cytoplasmic vacuoles. After treatment for 1 hour with 100 nmol/L of PMA at 37 degrees C, the cells assumed a spread-out, flasklike shape, displayed a low nuclear-to-cytoplasmic ratio, and exhibited cytoplasmic vacuoles. An inert but lipophilic phorbol diester, 4 phorbol 12,13-didecanoate, failed to induce these morphological changes. Cell kinetic studies showed that whereas SW 48 cells have a doubling time of 35 hours, those incubated with 100 nmol/L of PMA have a doubling time of 90 hours. Although the flow cytometry histograms were similar until 8 hours into the cell cycle, the PMA-treated cells ultimately spent proportionately less time in S and more in G2/M. Finally, under routine growth conditions, SW 48 cells express neither carcinoembryonic antigen nor G7 antigen. These antigens, which are present on the surface of well-differentiated cells, were expressed after treatment of SW 48 with PMA. The data suggest that PMA causes profound changes in structure, cell growth kinetics, and antigen expression, consistent with induction of differentiation of the cell line SW 48.

A pretreatment combination of physostigmine and azaprophen (6-methyl-6-azabicyclo[3.2.1]octan-3-ol-2,2-diphenylpropionate), a novel cholinolytic, was evaluated for its ability to minimize soman-induced incapacitation and lethality in guinea pigs. This was accomplished by using response surface methodology to model and analyze the combination, varying physostigmine from 0 to 194 micrograms/kg, azaprophen from 0 to 5 mg/kg, and soman from 30 to 150 micrograms/kg. One hundred percent survival was achieved against 5 LD50 of soman using as little as 100 micrograms/kg of physostigmine in the presence of 5 mg/kg azaprophen. Both survival and soman-induced incapacitation were similarly affected by this pretreatment combination. For both endpoints, greater efficacy was achieved with the combination than could be achieved with either component alone (therapeutic synergism). This suggests that such a pretreatment combination may prove very efficacious against soman-induced lethality and incapacitation in higher species.

Physostigmine (PHY) has the advantage over pyridostigmine of minimizing OP-induced incapacitation because it penetrates into the CNS. However, physostigmine is behaviorally toxic at relatively low concentrations. It is anticipated that this could be offset by a cholinolytic to prevent behavioral deficit due to the carbamate pretreatment alone. The therapeutic efficacy of physostigmine/azaprophen pretreatment therapy was evaluated in soman-challenged guinea pigs. Response surface methodology was employed to describe the relationship of the pretreatment combination with duration of incapacitation. The significance of the combination relative to PHY alone was evaluated in addition to dose combinations that yield optimal time to recovery. Analysis of the fitted response surface indicated that combination pretreatment with these compounds significantly reduces the time to recovery after soman challenge versus pretreatment with PHY alone.

Guinea pig alveolar cells were obtained in situ via bronchoalveolar lavage. The cells were 86% macrophages (GPAM), (greater than 97% viability) with the remainder of the population comprised of lymphocytes and eosinophils. The following battery of functional assays were studied in GPAM: chemotaxis was stimulated by N-formyl-methionyl-leucine-phenylalanine (FMLP) and by phorbol-12-myristate-13-acetate (PMA) in a concentration-related manner; cytotoxicity as measured by 51Cr release from target cells +/- PMA was induced in P815 mastocytoma cells and less strongly in 3T3 normal mouse fibroblasts; release of N-acetyl-beta-D-glucosaminidase (NAGA) was stimulated by the calcium ionophore A23187, but not by PMA or the combination of PMA + A23187; superoxide anion production as measured by the reduction of ferricytochrome C was stimulated 25-fold by PMA; phagocytosis of opsonized 51Cr sheep red blood cells occurred in a time-related manner and reached its maximum after 120 min; and cell spreading, which exhibited a high rate of spontaneous spreading (76%), was only minimally stimulatable by PMA. The responsiveness of the GPAM, the ease of retrieval, and the large numbers of cells available make the guinea pig an ideal system for future study.

The significance of the macrophage in the inflammatory response that occurs concurrently with phorbol ester induced tumor promotion has not yet been determined. Biologically active phorbol ester tumor promoters modify several functional responses of macrophages including chemotaxis, cytotoxicity, secretion and prostaglandin synthesis and release. The present study examines calcium metabolism as a possible underlying biochemical mechanism through which 12-0-tetradecanoyl-phorbol-13-acetate (TPA) exerts its effects on macrophage chemotaxis. The chemotaxis of mouse resident peritoneal macrophages was evaluated in the presence of pharmacological agents known to alter cellular calcium metabolism. The calcium ionophore A23187 in microM concentrations enhanced macrophage chemotaxis to TPA by approximately 41%. This enhancement was dependent on the presence of extracellular calcium. TPA-induced chemotaxis was also enhanced by the histological dye ruthenium red (RR), an agent known to modify mitochondrial calcium fluxes and calcium-dependent neuronal transmission. Ruthenium red (0.1 and 1.0 microM) produced a maximal stimulation of macrophage chemotaxis to TPA of approximately 62%. An intracellular calcium antagonist, 8-(N,N-diethylamino) octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) inhibited macrophage chemotaxis to TPA in a dose related fashion (1.0 to 100 microM). Varying extracellular calcium concentrations (0-3.6 mM) had no effect on macrophage chemotaxis in response to TPA. In drug combination studies neither A23187 nor RR was able to overcome the inhibitory effects of TMB-8 on macrophage chemotaxis to TPA. These results indicate that intracellular calcium metabolism may be playing a significant role in modulating TPA's effect on macrophage chemotaxis, while extracellular calcium may be of little import. A possible mode of TPA's effect on the macrophage via mobilization of calcium from cellular storage sites is discussed.

Sequential stimulation and washout procedures were employed to examine the kinetics and reversibility of pharmacologically manipulated second messenger signals mediating phenotypic changes and proliferative activation of resting human T lymphocytes. Phorbol dibutyrate (PDBu) was used to stimulate protein kinase C (Ca2+/phospholipid-dependent enzyme) while ionomycin was used to manipulate intracellular Ca2+ levels. Stimulation by PDBu alone induced phosphorylation of several endogenous substrates and altered expression of phenotypic markers, downregulating expression of CD4 and CD3 while increasing expression of CD2 and the interleukin 2 (IL-2) receptor. Stimulation with ionomycin alone caused an increase in intracellular Ca2+ levels but did not induce proliferation or cause major changes in the expression of phenotypic markers (CD2, CD3, CD4, CD8, IL-2, and transferrin receptors). Analysis of endogenous PDBu stimulated phosphosubstrates indicated that some substrates (pp92, pp82, pp55) underwent dephosphorylation, returning to base-line levels following PDBu removal while others (pp61, pp65) showed only partial dephosphorylation, while one (pp28) remained phosphorylated. Washing ionomycin-stimulated cells resulted in an approximately 75% reduction of intracellular Ca2+. Ionomycin exposure did not alter the affinity (KD = 22.3 +/- 7.4 nM) or number of receptors (53,497 +/- 8,291 receptors/cell) for [3H]PDBu. These data suggest that signals induced by PDBu or ionomycin are reversible following removal of the stimulating agents with respect to proliferative activation of T lymphocytes. Furthermore, a transcriptional mechanism regulating the production of IL-2 mRNA requires simultaneous activation of protein kinase C and elevation of intracellular Ca2+.

The B oligomer of pertussis toxin serves as a weak mitogen in the T lymphocyte, an effect which is associated with an early rise in cytosolic free calcium concentrations, as monitored by Fura-2 fluorescence. Upon co-administration of phorbol dibutyrate, a phorbol ester tumour promotor which activates protein kinase C, pertussis toxin-induced proliferation was synergistically enhanced, as measured by the increased uptake of [3H]thymidine, into cellular DNA. Although phorbol ester co-administration has often been associated with an inhibition of Ca2+-mobilizing pathways, phorbol dibutyrate pretreatment had no inhibitory effect on the pertussis toxin-induced calcium flux and may actually have enhanced this response slightly. Flow cytometric analysis of cell populations expanded by the combined regimen did not provide evidence for the preferential expansion of cells bearing either CD4 or CD8, the T-cell determinants representative of the helper-inducer and cytotoxic-suppressor subsets, respectively. Pertussis toxin and phorbol dibutyrate appear, therefore, to elicit polyclonal stimulation, rather than the selective activation of a given lymphocyte subset. Expression of the transferrin receptor, a marker for nutrient uptake, and CD25, the Tac component of the interleukin-2 (IL-2) receptor, was, however, synergistically enhanced in cells activated by the co-treatment procedure.

Several of the cannabinoids found in marihuana have been shown to inhibit tumor growth and increase the life-span of mice bearing the Lewis lung adenocarcinoma. When trypsin-dispersed isolated Lewis lung cells are incubated in vitro, they maintain their capacity to carry out macromolecular synthesis (RNA, DNA, protein). This process can be inhibited by cytosine arabinoside, actinomycin D, or methyl-1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, whereas cyclophosphamide, an agent that must be bioactivated, was inactive. Inhibition of DNA synthesis as measured by [3H]thymidine uptake into acid-insoluble material was used as an index of cannabinoid activity against isolated Lewis lung cells, L1210 leukemia cells, and bone marrow cells incubated in vitro delta9-, delta8-, 1-hydroxy-3-n pentyl-, and 1-delta8-tetrahydrocannabinol, and cannabinol demonstrated a dose-dependent inhibition of DNA synthesis whereas cannabidiol and 1-hydroxy-3-n-pentylcannabidiol were markedly less inhibitory in our in vitro cell systems. Furthermore, our in vitro observations with these cannabinoids are supported by in vivo tumor inhibition studies. Ring modifications as in cannabichromene or cannabicyclol abolish in vitro activity as does dihydroxylation at the 8beta and 11 positions of 1-delta9-trans-tetrahydrocannabinol. Delta9-trans-tetrahydrocannabinol demonstrated the least toxicity of all inhibitory cannabinoids in vivo; this is supported by its lesser effect on bone marrow DNA synthesis in vitro.

The neurotoxic organochlorine insecticide chlordecone (Kepone) was examined in several in vitro and in vivo neurochemical systems in an attempt to identify neurochemical alterations that might be relevant to the central nervous system manifestations of chlordecone toxicity in humans. In vitro, chlordecone was a remarkably potent inhibitor of brain mitochondrial oxidative phosphorylation and associated Ca2+ transport (Ki congruent to 10(-7) M). At a high concentration of chlordecone (10(-5) M), destabilization of biological membranes was observed. Both of these effects appeared to contribute to inhibition of synaptosomal Ca2+ uptake, which was accompanied by a pronounced, although paradoxical, stimulation of neurotransmitter release. Studies of the disposition of [14C]chlordecone revealed that the concentrations that elicited neurochemical changes in vitro were comparable to the brain tissue chlordecone concentrations achieved with a 40 mg/kg tremorigenic dose in intact animals. However, no neurochemical correlates of chlordecone toxicity were observed in studies of dopamine and norepinephrine turnover in chlordecone-intoxicated animals. These findings are discussed in relation to the development of neurochemical assays appropriate for investigating neurotoxic agents.

Macrophage spreading over glass surfaces is a recognized in vitro manifestation of activation. We examined macrophage spreading using a simplified assay. Hartley guinea pigs (n = 15) were anesthetized with pentobarbital (0.4 mg/gm) IM. Macrophages were obtained by lavaging the peritoneal (PM) and alveolar (AM) spaces with sterile 0.9% NaCl (NS). AM or PM (150,000 cells) were placed into chambers of Lab-Tek microtiter slides +/- phorbol myristate acetate (PMA). Slides were incubated /37 degrees C in 5% CO2 for 20 minutes. Macrophages with a diameter greater than 2 x control cells were considered spread. Compared to PM, resident AM show increased spontaneous spreading (16 +/- 2% vs 79 +/- 2%) respectively. AM demonstrated no significant concentration-dependent response to PMA stimulation. The PM has served as the basis for much of the morphological and functional observations attributed to macrophages in general. The above spreading data support the concept of disparity of macrophage function dependent upon various factors, including site of origin. Our observations suggest that extrapolation of macrophage characteristics to cells from discordant sources may not be possible.

Administration of cyclophosphamide (5, 10, 20 and 25 mg/kg body weight) to male CD-1 mice 2 hr after subcutaneous implantation of a 5-bromo-2'-deoxyuridine (BrdUrd) pellet (55 mg) resulted in a dose-dependent increase in sister chromatid exchanges (SCE) in bone marrow cells. Treatment with cyclophosphamide (15 mg/kg body weight) at the time of BrdUrd implantation and 2, 6.5, and 13 hr post-BrdUrd implantation resulted in the induction of approximately 19 SCE/cell indicating that the bone marrow SCE response was independent of the time of administration. Treatment with cyclophosphamide (15 mg/kg body weight) at 26, 19, 13, and 6 hr prior to BrdUrd implantation resulted in baseline SCE (3.3 SCE/cell) at 26 hr with an increasing number of SCE/cell with decreasing time prior to BrdUrd implantation. These results compare favorably with those obtained by Kram et al [1981] with mitomycin C (MMC) using a similar protocol. The time-dependent induction of SCE is qualitatively similar for CP and MMC, both of which are bifunctional alkylating agents metabolically activated by oxidation and reduction, respectively, and suggests that these two compounds may induce SCE by a similar mechanism.

ACTH, cholera toxin, cyclic AMP but not pregnenolone-induced steroidogenesis in Y-1 functional mouse adrenal tumor cells was significantly inhibited by delta-9-tetrahydrocannabinol, cannabidiol, and cannabinol. The inhibition of steroidogenesis could not be correlated with a general depression in cell function or viability. The data suggest that cannabinoids inhibit corticosteroidogenesis at a site between the synthesis of cAMP and of pregnenolone.

Biologically active phorbol ester derivatives displace [3H]phorbol-12, 13-dibutyrate from thioglycollate-elicited mouse peritoneal macrophages in a time-, temperature-, and concentration-dependent manner. Scatchard analysis revealed an apparent Kd of 54.1 nM and 8.0 X 10(5) sites/cell, indicating that these macrophages possess saturable, high-affinity phorbol ester-binding sites. These derivatives also act as chemoattractants for the macrophage at equivalent concentrations. A notable exception to this pattern is phorbol-12,13-diacetate. Phorbol-12,13-diacetate inhibits specific binding of [3H]phorbol-12,13-dibutyrate (concentration required for a 50% inhibition of the maximum specific binding of [3H]phorbol-12,13-dibutyrate, 2.6 microM) and chemotaxis to phorbol-12-myristate, 13-acetate (concentration required for a 50% inhibition of the maximum chemotactic response, 0.39 microM) while exhibiting no activity as a chemoattractant at concentrations up to 10(-5) M. The data indicate that phorbol-12,13-diacetate may be an antagonist for receptor-mediated chemotaxis to phorbol-12-myristate, 13-acetate in the macrophage.

The relative potencies of various prostaglandins were investigated in trypsin-dispersed cat adrenocortical cells. Prostacyclin proved to be the most potent steroidogenic prostaglandin, being 100-1000 times more potent than PGE2. This stimulant effect of prostacyclin was only partially dependent upon the presence of extracellular calcium and was associated with increased levels of cyclic AMP. These data suggest a possible role for prostacyclin in corticosteroidogenesis.

We found a dose-related decrease in DNA synthesis in transformed cell cultures treated with delta9-tetrahydrocannabinol (delta9-THC). The decrease, observed over a 4-hour period, was not accompanied by a change in the radioactive precursor pool as compared to that of control culture. The distribution of labeled products clearly differed from that observed after treatment with cytosine arabinoside. delta9-THC inhibited DNA synthesis at some point beyond the uptake of 3H-thymidine.

A program for the calculation of the median effective dose (ED50) and the slope of the dose-effect line was developed for a programmable calculator. The method employed approximated the solution described by Bliss. Experimental data were evaluated and compared to both hand calculated results and results of other computer methods. This method produced results which differed from other computer methods by less than 1 percent. This program provided information necessary for the test for parallelism and estimate of relative potency of two dose-effect lines.

The phorbol ester tumor promoter phorbol-12-myristate-13-acetate (PMA) induced mouse resident peritoneal macrophage spreading in an in vitro system in a time- and dose-dependent manner; this process was modified by agents which alter intracellular calcium metabolism. After a 35-min incubation with PMA, 50% of the macrophages were spread (as classified by at least a 2-fold increase in cell surface area). Also at 35 min, the median effective concentration for PMA induction of spreading was 1.6 ng/ml. The intracellular calcium antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate inhibited PMA-induced cell spreading with a one-half maximal inhibitory concentration of 8.0 microM. The calcium ionophore A23187 enhanced PMA-induced spreading by 30% at 0.1 to 10 nM. The histological dye ruthenium red, which purportedly increases intracellular calcium by displacing membrane-bound calcium stores, enhanced PMA-induced spreading up to 75% at 1.0 pM. The cationic chelator ethyleneglycolbis(beta-aminoethylether)-N,N-tetraacetic acid (1.8 and 3.6 mM) had no effect on PMA-induced spreading. Thus, PMA-induced spreading was independent of extracellular calcium but was modulated by agents altering intracellular calcium metabolism. Microfilament formation, a proposed mechanism of cell spreading, also depends on intracellular calcium availability. The microfilament inhibitor cytochalasin B inhibited PMA-induced spreading with a one-half maximal inhibitory concentration of 1 microM. Future experiments should investigate the hypothesis that calcium availability to the cytoskeletal elements regulates the morphological effects of PMA on macrophages.

Chinese hamster cells (V79) were treated with ethylnitrosourea (ENU) and cis-diamminedichloroplatinum(II) (DDP) alone and in combination. Sister chromatid exchanges (SCEs) were quantified as measures of genotoxicity of the two agents. The combination experiment employed a factorial design in which cells were treated, in various concentration combinations, with both agents simultaneously. Response surface methodology, using a polynomial model based on a negative binomial distribution of SCE events, was employed for analysis of the interactions of the two genotoxic agents. The negative binomial distribution, a generalization of the Poisson distribution, is required since SCEs are discrete variables which, under the conditions of these experiments, have a distribution which exhibits extra-Poisson variability. The model of the ENU/DDP combinations indicated an increasingly less-than-additive effect resulting from increasing concentrations of each agent in the combination. The analysis of these experiments demonstrates the usefulness of a powerful statistical procedure for evaluating the biological effects resulting from exposure to multiple cytotoxic agents. The methodology can be used with many other types of endpoints and is not limited by the number of treatment agents.

Transport of methotrexate in mammalian cells is a complex process. Although the drug is pumped uphill into cells, metabolic poisons enhance influx and transmembrane gradients for this agent. Structurally unrelated inorganic and organic anions nonspecifically depress these parameters in a variety of tumor cells. It has been suggested that cell cyclic adenosine 3':5'-Monophosphate (cyclic AMP) plays a regulatory role in the transport of this agent. To evaluate this further, a number of different experimental approaches were used to analyze the relationship between methotrexate transport and cell cyclic AMP in the Ehrlich ascites tumor. The following results indicate that for the Ehrlich ascites tumor, at least, there is no evidence for a regulatory role for cyclic AMP in the transport of methotrexate. (a) A marked increase in cell cyclic AMP by cholera-toxin or reduction in cyclic AMP by ascorbate was unaccompanied by changes in methotrexate influx. (b) Instantaneous and constant inhibition of methotrexate influx by isobutylmethylxanthine was temporally dissociated from the slower rise in cell cyclic AMP that was induced by this agent. (c) Although isobutylmethylxanthine and dibutyryl cyclic AMP augment cell cyclic AMP and decrease methotrexate influx, they have different effects on efflux and net transport of methotrexate. Isobutylmethylxanthine stimulates net methotrexate uptake and slows methotrexate efflux but dibutyryl cyclic AMP depresses net methotrexate uptake without an effect on methotrexate efflux. (d) Azide markedly stimulates methotrexate influx and net transport without a change in cell cyclic AMP. (d) Dinitrophenol inhibits methotrexate influx and augments net methotrexate accumulation but does not alter cell cyclic AMP.

Chemotaxis and lysosomal enzyme release in peripheral blood neutrophils taken from patients before, during, and after recovery from the adult respiratory distress syndrome (ARDS) were studied. This allowed for correlation of cellular function with changes in a patient's clinical status. It was found that neutrophils from 8 of 9 patients with the fully developed syndrome exhibited a profound depression of chemotaxis (63% depressed, P = 0.0001) and a fourfold elevation of basal lysosomal enzyme release relative to neutrophils from healthy controls (P = 0.0001). These findings of depression of chemotaxis and enhanced basal enzyme release were also detected in neutrophils taken from 7 of 11 patients in whom clinical risk factors (eg, sepsis, pneumonia) for the syndrome had developed. Following resolution of the adult respiratory distress syndrome, the above changes in neutrophil function resolved in the four patients studied during convalescence. Healthy neutrophils exposed to plasma samples (untreated or zymosan-activated) from control subjects and patients with ARDS could not be distinguished with respect to chemotaxis and enzyme secretion. It is concluded that patients in whom ARDS develops show profound but reversible changes in peripheral neutrophil activity which can be measured following the development of a clinical predisposition for the syndrome. Further, the presence of a humoral substance capable of promoting chemotaxis or enzyme secretion from healthy neutrophils in the untreated plasma of patients suffering from ARDS was not demonstrated. This suggests that alteration of neutrophil activity measured in patients with the fully developed syndrome may be cellular in origin.

The potent promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), markedly enhanced the ability of mouse peritoneal macrophages to inhibit the growth of L5178Y tumor cells as measured by growth in agar. Three populations of macrophages, resident, divinylether maleic anhydride copolymer, and thioglycollate-recruited, were used. In general, TPA reduced both the cocultivation time and the number of macrophages required to induce cytotoxicity in all three macrophage types. With divinylether maleic anhydride copolymer macrophages, TPA enhanced cytotoxicity in a dose-dependent manner in the concentration range of 1.7 to 170 nM at macrophage: tumor cell ratios of 10:1 and 1:1. For reasons that were not apparent, inhibition of cytotoxicity was found at higher cell ratios. With both thioglycollate-elicited and resident macrophages, TPA (170 nM) enhanced cytotoxicity at all ratios tested. Even 1:1 ratios of macrophages:tumor cells, which were not cytotoxic alone, inhibited cell viability by 50% to 60% in the presence of TPA. A correlation was found between the biological activity of related macrocyclic diterpenes and their ability to enhance macrophage-mediated cytotoxicity. Thus, mezerein and phorbol didecanoate enhanced macrophage cytotoxicity, while the biologically inactive analogs, phorbol, 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate, and 4-alpha-phorbol-12, 13-didecanoate were without effect in this assay. Cytotoxicity towards untransformed BALB/c/3T3 cells was also demonstrated using a liquid cloning assay. These target cells were much less sensitive to growth inhibition by the macrophages than were the L5178Y cells. A 50% decrease in survival occurred only after 48 hr incubation and required macrophage: target cell ratios of 100:1. The addition of 170 nM TPA led to a dramatic enhancement of cytotoxicity in these cells at macrophage:target cell ratios of 10:1 and 1:1. The results observed with the phorbol esters in the present studies are compatible with other evidence that these compounds can modulate a variety of macrophage functions.

Administration of ethylnitrosourea (ENU) (20, 25, 40, 50, and 60 mg/kg body weight) or methylnitrosourea (MNU) (25, 40, 50, 60, 75, and 80 mg/kg body weight) to male CD-1 mice 2 hr after sc implantation of a 5-bromo-2'-deoxyuridine (BrdUrd) pellet (55 mg) resulted in a dose-dependent increase in sister chromatid exchanges (SCE) in bone marrow cells. Treatment with ENU (50 mg/kg body weight) at several time points prior to BrdUrd implantation resulted in a multiphasic curve of SCE induction indicating at least two events that result in SCEs. Treatment with ENU at the time of BrdUrd implantation and post-BrdUrd reflected a similar mechanism apparent in the pre-BrdUrd curve. Treatment with MNU (50 mg/kg body weight) pre- and post-BrdUrd resulted in a linear monophasic curve of SCE induction in both the pre- and post-BrdUrd time periods. The overall MNU time-course curve resembled an inverted V function suggesting the mechanisms of SCE induction for ENU and MNU are different. These observations suggest that at least one explanation for the differences in the time courses for ENU and MNU SCE induction may result from a more persistent lesion being induced by ENU. In addition, these results indicate that in vivo SCE protocols which utilize a single acute chemical exposure at or near the time of BrdUrd labeling may not be useful for judging the relative activities of genotoxic agents.

The mutagenic potential of povidone-iodine (PVP-I) and some related compounds were studied using the L5178Y mouse (TK+/-) lymphoma assay. The established mutagens ethyl methanesulfonate (EMS) and dimethylnitrosamine (DMN) were highly active in this assay, whereas PVP-I, polyvinyl pyrolidone (PVP), potassium iodide (KI), and iodine (I2) were inactive. Furthermore, in the presence of a rat liver microsomal activating fraction (S-9), PVP-I and I2 had only marginal activity as mutagens. Using the Balb/c 3T3 transformation assay we assessed the transformational capacities of these same agents and the positive mutagen N-methyl-N-nitro-N-nitrosoguanidine (MNNG). All concentrations of the compounds tested were inactive in this assay except for PVP-I (5 mg/ml) and MNNG (5 micrograms/ml). However, the response with PVP-I was only marginal. We concluded from these studies that PVP, PVP-I, KI, and I2 did not possess any biologically significant mutagenic or cell transforming ability.

The ability of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), to induce chemotaxis in three different populations of mouse peritoneal macrophages was studied. TPA in the range of 10(-9) to 10(-7) M produced a dose- and time-related increase in chemotaxis in resident, thioglycollate-elicited, and divinyl ether maleic anhydride copolymer-activated macrophages. A maximal response was obtained after 4 hr incubation with 10(-7) M TPA, and this concentration of TPA was as effective as inducing chemotaxis as was endotoxin-activated mouse serum. Orientation of macrophages towards TPA was also observed by microscopy. Within 2 hr, cells exposed to TPA sent out cytoplasmic processes along the TPA gradient. Parallel arrays of cells oriented towards the TPA were observed after 4 hr incubation. Two other diterpene tumor promoters, phorbol-12,13-didecanoate and mezerein, were also chemotactic for the macrophages, as was the peptide epidermal growth factor, which shares a number of effects with TPA on cells in culture. On the other hand, two phorbol esters inactive as tumor promoters, 4-alpha-phorbol-12,13-didecanoate and phorbol, were not chemotactic for macrophages. Retinoic acid, which inhibits tumor promotion, inhibited TPA-induced, but not endotoxin-activated mouse serum-induced chemotaxis. These findings, taken together with previous studies, indicate that phorbol ester tumor promoters are potent modulators of macrophage function.