Effects of delta9-tetrahydrocannabinol in Lewis lung adenocarcinoma cells in tissue culture.

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.

Antineoplastic activity of cannabinoids.

Lewis lung adenocarcinoma growth was retarded by the oral administration of delta9-tetrahydrocannabinol (delta9-THC), delta8-tetrahydrocannabinol (delta8-THC), and cannabinol (CBN), but not cannabidiol (CBD). Animals treated for 10 consecutive days with delta9-THC, beginning the day after tumor implantation, demonstrated a dose-dependent action of retarded tumor growth. Mice treated for 20 consecutive days with delta8-THC and CBN had reduced primary tumor size. CBD showed no inhibitory effect on tumor growth at 14, 21, or 28 days. Delta9-THC, delta8-THC, and CBN increased the mean survival time (36% at 100 mg/kg, 25% at 200 mg/kg, and 27% at 50 mg/kg, respectively), whereas CBD did not. Delta9-THC administered orally daily until death in doses of 50, 100, or 200 mg/kg did not increase the life-spans of (C57BL/6 times DBA/2)F1 (BDF1) mice hosting the L1210 murine leukemia. However, delta9-THC administered daily for 10 days significantly inhibited Friend leukemia virus-induced splenomegaly by 71% at 200 mg/kg as compared to 90.2% for actinomycin D. Experiments with bone marrow and isolated Lewis lung cells incubated in vitro with delta9-THC and delta8-THC showed a dose-dependent (10(-4)-10(-7)) inhibition (80-20%, respectively) of tritiated thymidine and 14C-uridine uptake into these cells. CBD was active only in high concentrations (10(-4)).

Maleic vinyl ether activation of murine macrophages against lung-metastasizing tumors.

A 16,500 molecular weight fraction of maleic vinyl ether (MVE-2) induced tumoristatic and tumoricidal activity in peritoneal macrophages of BALB/c and C57BL/6 mice following i.p. administration. Growth of B16 melanoma cells in vitro was inhibited up to 85% by MVE-2-activated, but not resident, peritoneal macrophages. In a tritiated thymidine release assay, B16 melanoma cells, and to a lesser extent Madison 109 lung carcinoma cells, were also sensitive to the cytolytic action of MVE-2-activated peritoneal macrophages. Administration i.v. of MVE-2 resulted in tumoristatic and tumoricidal activity in alveolar macrophages against radiolabeled B16 and Madison 109 lung carcinoma target cells. MVE-2-activated alveolar macrophages significantly inhibited L5178Y lymphoma colony formation following a 48-hr macrophage-tumor cell coincubation. BALB/c mice bearing the lung-metastasizing Madison 109 lung carcinoma footpad tumor were given MVE-2 i.v., using the same dosing regimen that induced alveolar macrophages to be tumoricidal in vitro. Significant increases in life span were observed, suggesting that the antitumor activity of MVE-2 in this tumor system may be mediated by the activation of alveolar macrophages, with a resulting decrease in metastatic growth in the lung.

The inhibition of DNA synthesis by cannabinoids.

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.

Silicone-induced modulation of natural killer cell activity.

Current controversy regarding the effects of silicone gel mammary implants on the immune system has led to increased focus on the potential biological activity of silicone materials. Studies were undertaken in the B6C3F1 mouse to evaluate the immunotoxicological effects of the following components of a mammary gel prosthesis: polydimethylsiloxane fluid, silicone gel, elastomer shell and a polyurethane implant cover. Material was implanted subcutaneously (s.c.) for either 10 or 180 days. The only toxicological or immunological parameter which differed from control values was the natural killer (NK) cell activity in gel implanted mice. In these animals, basal NK activity was decreased. NK activity was comparably inhibited in female Fischer 344 rats implanted with silicone gel following short term (14 or 30 days) as well as chronic (1 year) exposure. The response was variable in both the rat and mouse. Host resistance to B16F10 melanoma in the mouse is known to be related to NK activity. Despite suppression of NK activity following gel implantation, host resistance was unaltered. Inhibition of NK activity by the administration of anti-asialo GM1 indicated that resistance was not adversely affected until NK activity was decreased by 40 to 50%. As with basal NK activity, augmented NK activity was inhibited in gel implanted rats when compared to controls. Administration of polyinosinic:polycytidylic acid to gel implanted rats resulted in an increase of approximately 65% in NK activity compared to gel implanted controls. This level of augmented NK activity was significantly less than the level of augmented NK activity in vehicle implanted animals where 100% enhancement in NK activity was noted. The results of these studies indicate that (1) NK cytolytic activity is altered in two animal models, the mouse and rat, although the results were variable, that (2) the level of suppression noted in the mouse is ineffective when compared to the level of suppression required to alter host resistance to B16F10 melanoma and that (3) the gel implanted animals exhibit a suppressed response to NK augmentation.

Suppression of the in vitro specific antibody response by 12-O-tetradecanoylphorbol-13-acetate (TPA) in the mouse.

The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), has several effects on the immune system. The effect of TPA on the in vitro antibody response to sheep red blood cells (sRBC) was studied. Spleen cells treated in vitro with TPA resulted in a dose-dependent inhibition of the IgM antibody forming cell (AFC) response with an IC50 of 0.74 nM. Nontumor promoting analogs at concentrations up to 1000 nM had no effect on this system. 2-Mercaptoethanol (2ME), which is known to augment macrophage function in this assay, did not reverse the inhibition. Spleen cells were separated into plastic adherent cells (ADC) and nonadherent cells (NAC). When NAC were treated with TPA and cultured with untreated ADC, the inhibition was seen. However, treating the ADC alone did not result in an inhibition. These results show that TPA will suppress the in vitro antibody response in the mouse and that the suppression is due to a selective effect on the plastic nonadherent cell population. The study of these effects of TPA may allow us to gain insight into the mechanism of its tumor promoting activity and to use it as a tool to study the modulation of the immune system.

Immunotoxic effects of diethylstilbestrol on host resistance: comparison with cyclophosphamide.

To evaluate the usefulness of host resistance assays for measurement of immunotoxicologic effects of chemicals, the immunosuppressive effects of exposure to diethylstilbestrol (DES) were compared with the effects of treatment with the known immunosuppressive drug cyclophosphamide (CPS). A panel of six host resistance models was evaluated, including infection with the bacterium Listeria monocytogenes, herpes simplex virus type 2 (HSV-2), and encephalomyocarditis virus (EMC), the yeast Cryptococcus neoformans, the parasite Naegleria fowleri, and transplantation of the B16F10 melanoma tumor. The results demonstrate a general correlation between the effects of CPS and DES on host resistance. Acute treatment with CPS (200 mg/kg) markedly depressed resistance to the microbial infections with L. monocytogenes and HSV, and exposure to DES usually also decreased resistance in a dose dependent manner. Moreover, CPS had no marked effect on resistance to N. fowleri and EMC virus, and exposure to DES also had a neglible or slight effect. There were, however, two model systems in which the effects of CPS and DES diverged. Whereas treatment with DES produced no significant effect on resistance to C. neoformans, acute treatment with CPS prior to the fungal infection produced a marked increase in resistance. Also, while treatment with CPS markedly increased B16F10 lung metastases, treatment with DES significantly decreased the incidence and number of lung metastases. The data support the general validity of host resistance assays, particularly with models of short disease course, for measuring immunosuppression. However, the results also emphasize the complexity of interpreting effects of environmental chemicals on host resistance, because of the interplay of such factors as relative times of exposure to the chemical in relation to pathogenesis of infection, the length of the disease course, the nature of the operative host defense mechanisms, and the compensatory recovery of these mechanisms.

In vitro immunotoxicological assays for detection of compounds requiring metabolic activation.

A system for metabolic activation of cyclophosphamide (CP), consisting of a crude microsomal fraction of mouse liver and necessary cofactors (S9 mix), was interfaced with three murine cell culture assays for immunotoxicity. These assays were: the Mishell-Dutton assay for in vitro antibody formation, splenic lymphocyte responsiveness to mitogens and bone marrow cell cultures. There was no effect of CP at doses up to 261 microgram/ml (lmM) on any of the parameters measured unless S9 mix was included. Much greater potency was achieved if the S9 mix was prepared from livers of mice pretreated with phenobarbital. Under these conditions and dose-related inhibition of plaque-forming cells (PFC) in the Mishell-Dutton assay was observed, yielding an ED50 of 6.3 microgram/ml. When splenic lymphocytes were exposed to CP in the presence of induced S9 mix, a dose related inhibition of the response to the B-cell mitogen, lipopolysaccharide (LPS), and to the T-cell mitogen, concanavalin A (Con A), was observed. For the optimum LPS concentration, the ED50 for CP was 8.1 microgram/ml; for the optimum concentration of Con A, the ED50 was 6.7 microgram/ml. DNA synthesis was not inhibited by the doses used. When bone marrow cells were exposed to CP in the presence of induced S9 mix, the stem cell population, enumerated by colonization in semisolid medium, was reduced in a dose-dependent manner, with an ED50 of 5.2 microgram/ml. Again, DNA synthesis was not affected unless higher doses of CP were used.

Toxicology of organic drinking water contaminants: trichloromethane, bromodichloromethane, dibromochloromethane and tribromomethane.

This study evaluated the subchronic toxicity of selected halomethanes which are drinking water contaminants. The compounds studied were trichloromethane, bromodichloromethane, dibromochloromethane and tribromomethane. Subchronic 14-day gavage studies were performed with the use of doses encompassing one-tenth the LD50 for the compounds. A 90-day gavage study of one of the compounds, trichloromethane, was also done. Parameters observed included body and organ weights, histopathology, hematology, clinical chemistries, and hepatic microsomal enzyme activities. Toxicity to the humoral immune system was assessed by measuring the number of splenic IgM antibody-forming cells and the serum antibody level to sheep erythrocytes. Cell-mediated immunity was evaluated by measuring the delayed type hypersensitivity response and popliteal lymph node proliferation response to sheep red blood cells. The functional activity of the reticuloendothelial system, as measured by the vascular clearance rate and tissue uptake of 51Cr sheep red blood cells was also determined. The major effects of the halomethanes were increased liver weights, elevations of SGPT and SGOT, decreased spleen weights and a decrease in the number of splenic IgM antibody-forming cells. The humoral immune system appeared to be an indicator of halomethane toxicity. There is evidence that subchronic 14-day exposure may be of greater value than long-term studies in determining the toxicity of these compounds.

In vivo assessment of immunotoxicity.

The organs, tissues, and cells of the lymphoreticular system have received considerable attention as targets for chemicals causing adverse effects. A basic toxicological approach is described for assessing the risk of a chemical perturbing the immune system. CD-1 mice were exposed for 14 or 90 days to one of several chlorinated hydrocarbons: 1,2-dichloroethane, 1,2-dichloroethylene or 1,1,2-trichloroethylene. Other mice were exposed to dexamethasone, a known immunosuppressive agent. The immune system is evaluated against a background of the more standard toxicological parameters such as fluid consumption, body and organ weights, hematology, clinical chemistries, and blood coagulation. Reported here are the results for the male mice after 14-day exposure to three chlorinated hydrocarbons and after 90-day exposure to 1,2-dichloroethane and dexamethasone.Acute toxicity studies were performed to provide a basis for doses used in the subchronic studies. The LD(50) values are reported. The status of the humoral immune system was determined by measuring the number of IgM spleen antibody-forming cells to sRBC, the serum antibody level to sRBC, and the lymphocyte response to the B-cell mitogen, LPS. Of the three chlorinated hydrocarbons, only dichloroethane produced a significant (p < 0.05) reduction in antibody-forming cells. The other two chemicals produced trends towards suppression. Mice exposed to dichloroethane in the drinking water for 90 days showed no alteration in AFC, serum antibody titers or response to the B-lymphocyte mitogen, LPS. Subchronic 90-day exposure to dexamethasone produced a dose-dependent inhibition of AFC/spleen but not AFC/10(6) spleen cells when measured on the peak day of response. Response to LPS was not altered, and spleen weight and spleen cell number were reduced as much as 42%. These data suggest that dexamethasone administered in the drinking water is nonspecifically cytotoxic to the spleen cells.Cell-mediated immunity was assessed by measuring the DTH response to sRBC and the response to the T-lymphocyte mitogen, concanavalin A. After 14 days of exposure, trichloroethylene produced a 15 and 60% suppression at 24 and 240 mg/kg, respectively. Dichloroethylene produced a non-dose-dependent inhibition at 4.9 and 49 mg/kg, which was slight, but significant (p < 0.05). Subchronic 90-day exposure to dichloroethane did not alter the DTH response or spleen lymphocyte response to concanavalin A. In contrast, dexamethasone produced a dose-dependent inhibition of the DTH response and a hyperresponsiveness to concanavalin A.Dichloroethane did not alter the functional activity of the reticuloendothelial system, as measured by the vascular clearance rate and tissue uptake of (51)Cr sRBC. In the case of dexamethasone exposure, only the spleen and thymus showed decreased uptake of (51)Cr sRBC, which was directly related to decrease in size. The approaches and results from these types of studies provide a basis for judging a chemical's potential risk to the immune system.