A method for screening groundwater vulnerability from subsurface hydrocarbon extraction practices.

This paper describes a new screening method for assessing groundwater vulnerability to pollution from hydrocarbon exploitation in the subsurface. The method can be used for various hydrocarbon energy sources, including conventional oil and gas, shale gas and oil, coal bed methane and underground coal gasification. Intrinsic vulnerability of potential receptors is assessed at any particular location by identifying possible geological pathways for contaminant transport. This is followed by an assessment of specific vulnerability which takes into account the nature of the subsurface hydrocarbon activity and driving heads. A confidence rating is attached to each parameter in the assessment to provide an indication of the confidence in the screening. Risk categories and associated confidence ratings are designed to aid in environmental decision making, regulation and management, highlighting where additional information is required. The method is demonstrated for conventional gas and proposed shale gas operations in northern England but can be adapted for use in any geological or hydrogeological setting and for other subsurface activities.

Interleukin 1-induced, T cell-mediated regression of immunogenic murine tumors. Requirement for an adequate level of already acquired host concomitant immunity.

Intraperitoneal injection of human rIL-1 in a dose of 0.5 microgram daily for 5 d, or 1 microgram daily for 3 d, was capable of causing complete regression of immunogenic SA1 sarcoma growing subcutaneously in syngeneic or semisyngeneic mice. Higher doses of IL-1 were not more therapeutic against the SA1 sarcoma, but needed to be given to cause complete regression of the immunogenic L5178Y lymphoma. On the other hand, the P815 mastocytoma was much less responsive to IL-1 therapy, in that it failed to undergo complete regression in response to doses of IL-1 capable of causing regression of the L5178Y lymphoma. IL-1 caused regression of the SA1 sarcoma when given on days 6-8 of tumor growth, but not when given on days 1-3. This refractoriness of a small tumor to IL-1 therapy suggests that the antitumor action of IL-1 is based on an underlying host-immune response that is not generated until after day 3 of tumor growth. Direct evidence for the participation of host immunity in IL-1-induced tumor regression was supplied by results showing that IL-1 was not therapeutic against the SA1 sarcoma growing in T cell-deficient (TXB) mice, unless these mice were first infused with Ly-2+ and L3T4+ T cells from donor mice bearing an established SA1 sarcoma. In contrast, normal T cells, or T cells from donor mice bearing a YAC-1 lymphoma, failed to provide TXB recipients with the ability to cause regression of their SA-1 sarcoma in response to IL-1 treatment. The results are in keeping with the interpretation that exogenous IL-1, by augmenting the production of tumor-sensitized T cells, converts a subtherapeutic level of host immunity to a therapeutic level. The results suggest, in addition, that IL-1 only stimulates the replication of T cells that are already engaged in the antitumor immune response.

Potency values from the local lymph node assay: application to classification, labelling and risk assessment.

Hundreds of chemicals are contact allergens but there remains a need to identify and characterise accurately skin sensitising hazards. The purpose of this review was fourfold. First, when using the local lymph node assay (LLNA), consider whether an exposure concentration (EC3 value) lower than 100% can be defined and used as a threshold criterion for classification and labelling. Second, is there any reason to revise the recommendation of a previous ECETOC Task Force regarding specific EC3 values used for sub-categorisation of substances based upon potency? Third, what recommendations can be made regarding classification and labelling of preparations under GHS? Finally, consider how to integrate LLNA data into risk assessment and provide a rationale for using concentration responses and corresponding no-effect concentrations. Although skin sensitising chemicals having high EC3 values may represent only relatively low risks to humans, it is not possible currently to define an EC3 value below 100% that would serve as an appropriate threshold for classification and labelling. The conclusion drawn from reviewing the use of distinct categories for characterising contact allergens was that the most appropriate, science-based classification of contact allergens according to potency is one in which four sub-categories are identified: 'extreme', 'strong', 'moderate' and 'weak'. Since draining lymph node cell proliferation is related causally and quantitatively to potency, LLNA EC3 values are recommended for determination of a no expected sensitisation induction level that represents the first step in quantitative risk assessment.

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.

Mutagenic activity of tumor-associated macrophages in Salmonella typhimurium strains TA98 and TA 100.

Suspensions of cells from a series of strain BALB/cfC3H mouse mammary tumors, and adherent and nonadherent cells from the tumors, were tested for their ability to increase the mutation rate of Salmonella typhimurium tester strains TA98 and TA100. Significant increases were seen with cells from three of four tumor lines tested on the TA98 strain and with one of four tested on the TA100 strain. The mutagenic activity was due primarily to cells in the adherent fractions which were greatly enriched for macrophages.

Cytokine mRNA profiles for isocyanates with known and unknown potential to induce respiratory sensitization.

Isocyanates are low-molecular-weight chemicals implicated in allergic asthmatic-type reactions. Identification of chemicals likely to cause asthma is difficult due to the lack of a validated test method. One hypothesis is that differential cytokine induction (Th1 versus Th2 profiles) in the draining lymph node following dermal application can be used to identify asthmagens and distinguish them from contact allergens. In this study, we compared the cytokine mRNA profiles of six chemicals: toluene diisocyanate (TDI), diphenylmethane-4,4'-diisocyanate (MDI), dicyclohexylmethane-4,4'-diisocyanate (HMDI), isophorone diisocyanate (IPDI), p-tolyl(mono)isocyanate (TMI), and meta-tetramethylene xylene diisocyanate (TMXDI). Whereas TDI and MDI are well-known respiratory sensitizers, documentation for HMDI, IPDI, TMI, and TMXDI is limited, but suggests that HMDI and IPDI may have respiratory sensitization potential in humans and TMI and TMXDI do not. Following dermal exposure of BALB/c mice, all six isocyanates induced cytokines characteristic of a Th2 response. Although LLNAs suggested that the doses chosen for the RPA were immunologically equivalent, the isocyanates tested differentiated into two groups, high responders and low responders. However, two of the low responders (TMI and TMXDI) were further tested and induced higher levels of Th2 cytokine message than dinitrochlorobenzene (not an asthmagen). Further study of these chemicals is needed to determine whether the Th2 cytokine responses observed for these low responders is predictive of asthmagenic potential or represents an insufficient signal.

Evaluation of the primary humoral immune response following exposure of male rats to 17beta-estradiol or flutamide for 15 days.

There is a concern that certain industrial chemicals found in the environment may mimic or antagonize endogenous hormones and adversely affect the endocrine as well as the immune system. The objective of this study was to determine if exposure of Crl:CD (SD)BR male rats to 17beta-estradiol (17beta-E2), an estrogen receptor agonist, or flutamide (FLUT), an androgen receptor antagonist, would significantly alter the primary IgM humoral immune response to sheep red blood cells (SRBC). This study was conducted in the context of a male in vivo Tier I battery designed to identify endocrine-active compounds (EACs). The Tier I male battery consists of organ weights coupled with a comprehensive hormonal assessment. Rats were dosed by the intraperitoneal route for 15 days with vehicle or 0.001, 0.0025, 0.0075, or 0.050 mg/kg/day 17beta-E2 or 0.25, 1, 5, or 20 mg/kg/day FLUT. Six days prior to termination, selected rats were injected intravenously with SRBC for assessment of humoral immune function. Spleen cell number and spleen and thymus weights were obtained. Serum was analyzed for anti-SRBC IgM antibody by using an enzyme-linked immunosorbent assay. At 0.050 mg/kg/day 17beta-E2, mean final body and absolute thymus weights were significantly decreased to 84 and 65% of control, respectively. 17beta-E2 did not significantly alter spleen weight, spleen cell number, or the primary IgM humoral immune response to SRBC. The no-observed-adverse-effect level (NOAEL) for immune system alteration was 0.050 mg/kg/day 17beta-E2 since the decrease in absolute thymus weight was judged to be secondary to the decrements in body weight. In the Tier I male battery, responses to 17beta-E2 included decreased absolute testis and epididymis weights, decreased relative accessory sex gland unit weights, hormonal alterations (decreased serum testosterone (T), dihydrotestosterone (DHT), and luteinizing hormone (LH), and increased serum prolactin and E2 levels). The lowest-observed-adverse-effect level (LOAEL) for the reproductive indices was 0.001 mg/kg/day 17beta-E2 based on the hormonal alterations seen at this level; no NOAEL was established. Exposure to FLUT did not significantly alter mean final body, spleen, or absolute thymus weights, spleen cell number, or the primary IgM humoral immune response to SRBC. A significant increase (118% of control) in relative thymus weight was observed at 20 mg/kg/day FLUT. The NOAEL for immune system alteration was 5 mg/kg/day FLUT based on the increased relative thymus weights that were judged to be compound-related. In the Tier I male battery, responses to FLUT included decreased absolute epididymis and relative accessory sex gland unit weights and hormonal alterations (increased serum T, DHT, E2, and LH, and decreased follicle stimulating hormone levels). The LOAEL for the reproductive indices was 0.25 mg/kg/day FLUT based on the hormonal alterations seen at this level; no NOAEL was established. Based on these data, the reproductive and not the immune system appears to be the primary target organ of toxicity in young adult male rats treated with either 17beta-E2 or FLUT.

Possible incorporation of an immunotoxicological functional assay for assessing humoral immunity for hazard identification purposes in rats on standard toxicology study.

The objective of this study was to examine the feasibility of conducting an immunotoxicological assay for assessing humoral immunity in rats on standard toxicology study. Male CD rats were untreated or dosed intraperitoneally daily for 30 or 90 days, excluding weekends, with vehicle or 2 mg/kg cyclophosphamide (CY). Six days prior to sacrifice, selected rats were injected intravenously with sheep red blood cells (SRBC). One day prior to necropsy, blood samples for hematological and clinical chemical measurements were collected from each rat. On the day of necropsy standard protocol tissues were collected, weighed, processed to slides, and examined microscopically. One-half of each spleen was used to prepare a single cell suspension in order to assess spleen cell numbers. Serum was analyzed for anti-SRBC IgM antibody using an enzyme-linked immunosorbent assay. A second set of studies was performed to examine further the effect of SRBC administration on lymphoid organ weights using 30- and 90-day study age-equivalent naive male CD rats. Exposure of animals to 2 mg/kg CY for 30 or 90 days resulted in a 28% and 61% decrease, respectively, in SRBC-specific serum IgM levels. CY treatment also caused mild alterations in some leukocytic parameters, with significant decreases of 35% and 33% in white blood cell and lymphocyte counts, respectively, observed in 30-day CY-treated animals receiving SRBC. Injection of SRBC alone did not alter hematological or clinical chemistry parameters. With the expected exception of the spleen (increased number and size of germinal centers), administration of SRBC did not significantly alter the weights or morphology of routine protocol tissues. Furthermore, administration of SRBC did not mask the immunosuppressive effects of CY treatment under the conditions of this study. Based on our preliminary findings, a functional assay for assessing humoral immunity may be conducted in animals on standard toxicology study.

Further evaluation of the incorporation of an immunotoxicological functional assay for assessing humoral immunity for hazard identification purposes in rats in a standard toxicology study.

A previous study (Ladics et al., 1995) conducted in our laboratory using the known immunosuppressant agent, cyclophosphamide, indicated that a functional assay for assessment of humoral immunity may be conducted in rats in a standard toxicology study. The objective of this study was to further examine the feasibility of conducting an immunotoxicological assay for assessing humoral immunity in rats in a standard toxicology study using a chemical, carbon tetrachloride (CCl4), whose principal target organ of toxicity is not the immune system. Specifically, the previous study and this study were done to determine whether the conduct of an assay for assessing humoral immune function would affect standard toxicological endpoints. Male CD rats were untreated or dosed orally for 30 or 90 days, excluding weekends, with vehicle or 12.5 or 25 mg/kg CCl4. Six days prior to sacrifice, selected rats were injected intravenously with sheep red blood cells (SRBC) for assessment of humoral immune function. One day prior to necropsy, blood for hematological and clinical chemical measurements was collected from each rat. On the day of necropsy standard protocol tissues were collected, weighed, processed to slides, and later examined microscopically. One-half of each spleen was used to assess spleen cell numbers and quantitate lymphocyte subsets (Thelper; Tcyt/sup; total T- and B-cells) by flow cytometry. Serum was analyzed for anti-SRBC IgM antibody by using an enzyme-linked immunosorbent assay. Administration of 12.5 and 25 mg/kg CCl4 for 30 days decreased SRBC-specific serum IgM levels 42 and 45%, respectively, while 25 mg/kg CCl4 for 90 days increased SRBC-specific IgM levels by 50%. CCl4 did not alter splenic lymphocyte subset numbers nor the weight nor morphology of lymphoid organs. Exposure to 25 mg/kg CCl4 did increase liver weight and serum sorbitol dehydrogenase levels, as well as produce centrilobular fatty change. SRBC administration did not alter any hematological or clinical chemistry parameters, nor lymphocyte subset numbers. With the expected exception of the spleen (slight increase in number and size of germinal centers), administration of SRBC did not significantly alter the weights or morphology of routine protocol tissues. Furthermore, administration of SRBC did not mask the rather mild hepatotoxic effects of CCL4 exposure observed in this study. Based on these and previous findings, it appears that a functional assay for assessing humoral immunity may be conducted in animals on standard toxicology study without altering standard toxicological endpoints.

An international evaluation of the murine local lymph node assay and comparison of modified procedures.

The murine local lymph node assay is a predictive test for the identification of skin-sensitizing chemicals. The method has been the subject both of national inter-laboratory studies and of extensive comparisons with guinea pig tests. In the investigations reported here, the local lymph node assay has been evaluated further in the context of an international study comprising five independent laboratories. In addition, the influence of minor modifications to the standard assay procedure on the performance of the test has been examined. The modified procedures investigated were exposure of mice for 4 rather than 3 consecutive days, excision of lymph nodes 4 rather than 5 days after the initiation of exposure and the use of an alternative isotope. All five laboratories, irrespective of whether the standard or a modified protocol was used, were able to identify accurately, and with comparable sensitivity, potassium dichromate and 2,4-dinitrochlorobenzene as skin sensitizers. Using standard criteria, none of the laboratories recorded positive responses with methyl salicylate, a non-sensitizer. In the standard protocol, lymph nodes are pooled for each experimental group and the vigor of responses measured as a stimulation index relative to vehicle controls. A stimulation index of 3 or greater is considered to indicate skin-sensitizing potential. One further modification adopted by three of the laboratories was to analyze nodes from individual animals and, thereby, permit statistical evaluation. This allowed a direct comparison of statistical significance with the conventional stimulation index as criteria for a positive response. The data indicate that, while statistical evaluation may provide, in some instances, for small increases in sensitivity, this may be at the expense of some loss of selectivity. There are, however, insufficient data presently to draw firm conclusions regarding the relative value of statistical analysis. These studies demonstrate that the local lymph node assay is sufficiently robust to accommodate minor procedural and technical modifications without material changes in test performance.

Further evaluation of the local lymph node assay in the final phase of an international collaborative trial.

The local lymph node assay (LLNA) is a method used for the prospective identification in mice of chemicals that have the potential to cause skin sensitization. We report here the results of the second and final phase of an international trial in which the performance of the assay has been evaluated using seven test materials in five independent laboratories. The additional chemicals examined here included compounds which are considered less potent allergens than some of those tested in the first phase of the investigation, and includes hexylcinnamic aldehyde (HCA), a chemical recommended by the Organization for Economic Cooperation and Development (OECD) as a positive control for skin sensitization studies. In each laboratory all skin sensitizing chemicals examined (2,4-dinitrochlorobenzene {DNCB}, HCA, oxazolone, isoeugenal and eugenol) elicited positive responses of comparable magnitude as judged by the derived lowest concentration of test chemical required to elicit a 3-fold or greater increase in the proliferative activity of draining lymph node cells compared with vehicle-treated controls. We observed that sodium lauryl sulphate, considered to be a non-sensitizing skin irritant, also induced a positive response in the assay. Para-aminobenzoic acid (pABA), a nonsensitizing chemical, was negative at all test concentrations in each laboratory. Some laboratories incorporated minor modifications into the standard assay procedure, including the evaluation of lymph nodes pooled from individual mice rather than treatment groups and the use of statistical analyses. The use of statistics did not markedly change the determination of the lowest concentration yielding a positive response. These data confirm that the local lymph node assay is robust and yields equivalent results when performed independently.

Identifying airway sensitizers: cytokine mRNA profiles induced by various anhydrides.

Exposure to low molecular weight (LMW) chemicals in the workplace has been linked to a variety of respiratory effects. Within the LMW chemicals, one of the major classes involved in these effects are the acid anhydrides. The immunological basis of respiratory hypersensitivity involves CD4+ cells. By virtue of their induction of cytokines typical of CD4+ T-helper type 2 (Th2) cells-interleukin (IL)-4, 10, and 13-respiratory sensitizers may be identified and differentiated from contact sensitizers which induce Th1 cytokines (IL-2 and IFN-gamma). Our previous work suggested that the ribonuclease protection assay (RPA) was useful in identifying the respiratory sensitizer, trimellitic anhydride (TMA), based on quantitative differences in Th2 cytokine mRNA as compared to the contact sensitizer dinitrochlorobenzene (DNCB). Therefore, the purpose of the studies described in this report was to expand the chemicals tested in the RPA. To this end, four acid anhydrides with known respiratory sensitization potential, TMA, maleic anhydride (MA), phthalic anhydride (PA) and hexahydrophthalic anhydride (HHPA), were tested. Although previously determined to induce immunologically equivalent responses in a local lymph node assay (LLNA), the initial dose chosen (2.5%) failed to induce Th2 cytokine mRNA expression. To determine if the lack of cytokine expression was related to dose, LLNAs were conducted at higher doses for each of the anhydrides. The highest doses evaluated (four- to six-fold higher than those used in the initial RPA) gave equivalent proliferative responses for the various anhydrides and were used for subsequent RPA testing. At these higher doses, significant increases in Th2 versus Th1 cytokine mRNA were observed for all anhydrides tested. These results suggest that the RPA has the potential to serve as a screen for the detection of LMW airway sensitizing chemicals. However, the basis for selecting immunologically equivalent doses may require some modification.

The local lymph node assay: a viable alternative to currently accepted skin sensitization tests.

The prospective identification of skin sensitizing chemicals is a vital prerequisite for their proper risk management. Traditionally this has been achieved largely by the conduct of guinea pig assays such as the maximization and Buehler tests. These methods are recommended by the Organisation for Economic Cooperation and Development (OECD) and are required by the European Union (EU) for the evaluation of new substances. However, a novel mechanistically based method, the local lymph node assay (LLNA), has been the focus of substantial validation activity in recent years. This material is reviewed in this paper. It is shown that the LLNA has been validated successfully by five interlaboratory assessments as well as by comparisons with guinea pig tests and human data. The method also offers clear advantages to the user in terms of objectivity, time and cost, and delivers important animal welfare benefits. In consequence, it is recommended that the LLNA be formally adopted by the OECD in Guideline 406 and accepted by the EU and US EPA as a method suitable for the classification of the skin sensitizing potential of chemicals.

Pathology considerations for, and subsequent risk assessment of, chemicals identified as immunosuppressive in routine toxicology.

Several proposals have been made with the aim of assisting in the early identification of chemicals with immunotoxic potential. The Organisation for Economic Cooperation and Development is now likely to incorporate enhanced immunopathology into the test guideline for the 28-day rat study, which may be regarded as a Tier I investigation. However, no guidelines have yet been proposed either for how the new data generated will be evaluated, or for how a subsequent risk assessment will be made. In this paper, considerations for the immunopathological assessment of the thymus, spleen, lymph nodes and bone marrow are described, together with comments on haematological and organ weight changes that may be associated with immunotoxicity. Their interpretation will depend on the doses at which changes are manifest, the quantity and quality of the effects observed and the presence and severity of other forms of toxicity. Lastly, risk assessment and the approach to Tier II testing in immunotoxicity is discussed. It is concluded that much of this work must be on a case-by-case basis, but should not in principle differ from the approach adopted for any other type of toxicity identified ina 28-day study.

The identification of chemicals with sensitizing or immunosuppressive properties in routine toxicology.

In the context of this paper, immunotoxicity is taken to encompass immunosuppression/immunopotentiation and allergy. Over the last 10 to 15 years, well characterized methods for the assessment of altered immune competence have been reported. This has led to proposals for tiered testing schemes. This review examines the suitability of immunotoxicity parameters for inclusion in routine 28-day studies and comments on methods that have been proposed for incorporation within the guidelines issued by the US FDA and US EPA and OECD. It is recommended that the existing OECD Guideline 407 is modified to incorporate total and differential blood cell counts, spleen and thymus weight and histopathology, and draining and distal lymph node histopathology for Tier I level testing. Data so generated will provide a reliable and accurate means of identifying at an early stage potential immunotoxic effects. Tier II testing should be carried out on a case by case basis and only assuming positive results are obtained at Tier I. An increasingly sophisticated understanding of the nature of immune responses to chemical allergens has facilitated the design of novel predictive methods for the identification of sensitizing activity. Opportunities which arise from these new developments in allergy testing such as the local lymph node assay, mouse ear swelling test, and the mouse IgE test should be monitored closely.

Assessment of the skin sensitization potential of topical medicaments using the local lymph node assay: an interlaboratory evaluation.

The murine local lymph node assay (LLNA) is a method for the predictive identification of chemicals that have a potential to cause skin sensitization. Activity is measured as a function of lymph node cell (LNC) proliferative responses stimulated by topical application of test chemicals. Those chemicals that induce a threefold or greater increase in LNC proliferation compared with concurrent vehicle controls are classified as skin sensitizers. In the present investigations we have evaluated further the reliability and accuracy of the LLNA. In the context of an international interlaboratory trial the sensitization potentials of six materials with a history of use in topical medicaments have been evaluated: benzoyl peroxide, hydroquinone, penicillin G, streptomycin sulfate, ethylenediamine dihydrochloride, and methyl salicylate. Each chemical was analyzed in the LLNA by all five laboratories. Either the standard LLNA protocol or minor modifications of it were used. Benzoyl peroxide and hydroquinone, both human contact allergens, elicited strong LLNA responses in each laboratory. Penicillin G, another material shown previously to cause allergic contact dermatitis in humans, was also positive in all laboratories. Streptomycin sulfate induced equivocal responses, in that this material provoked a positive LLNA response in only one of the five laboratories, and then only at the highest concentration tested. Ethylenediamine dihydrochloride dissolved in a 3:1 mixture of acetone with water, or in 4:1 acetone:olive oil (one laboratory), was uniformly negative. However, limited further testing with the free base of ethylene diamine yielded a positive LLNA response when applied in acetone:olive oil (AOO). Finally, methyl salicylate, a nonsensitizing skin irritant, was negative at all test concentrations in each laboratory. Collectively these data serve to confirm that the local lymph node assay is sufficiently robust to yield equivalent results when performed independently in separate laboratories and indicate also that the LLNA is of value in assessing the skin sensitization potential of topical medicaments.

Tumor-associated macrophages of mouse mammary tumors. I. Differential cytotoxicity of macrophages from metastatic and nonmetastatic tumors.

Macrophages were isolated from a series of transplanted mouse mammary tumors, originally derived from a single spontaneously arising tumor, to determine whether macrophage content or function correlated with any of a wide range of tumor properties. None of the tumor cell lines differed significantly in susceptibility to killing by MVE-2-activated peritoneal macrophages (cytotoxicity ranged from 40 to 63%). No significant differences were observed in macrophage content among the five tumor lines, nor was there any correlation between macrophage content and tumor weight, time since transplantation, or ability to metastasize. A significant association was observed, however, between a tumor's ability to metastasize to lung spontaneously and the tumoricidal activity of that tumor's infiltrating macrophages. Significant tumoricidal activity was seen when macrophages isolated from every metastatic tumor studied were used, whereas macrophage-mediated tumoricidal activity was observed in only 35% (6 of 17) of nonmetastatic tumors. Macrophage cytotoxicity was associated with spontaneous metastasis and not with lung colony formation per se.

Biodistribution and pharmacokinetics of recombinant, human 125I-interleukin-2 in mice.

The pharmacokinetics and biodistribution of radioiodinated recombinant interleukin-2 (125I-IL-2) was studied after either intravenous (i.v.) or intraperitoneal (i.p.) injection into C57BL/6 mice. Beta-lactoglobulin radiolabeled with 131I served as a control protein. After i.v. injection, 125I-IL-2 preferentially accumulated in the liver and spleen. Liver accumulation was fast, peaking at 5 min, and was followed by rapid clearance. Spleen accumulation was slightly slower, peaking at 15 min. Blood values 1 min after i.v. injection were 22-34% of the injected doses (I.D.)/gram. These values declined quickly over the next hour. In contrast, after i.p. administration no organ showed specific uptake of 125I-IL-2. Blood values after i.p. injection were essentially constant over 3 h and were greater and more sustained than after i.v. administration. Kidney values for both 125I-IL-2 and 131I-beta-lactoglobulin, after either i.v. or i.p. injection, indicated that the major route of clearance for both compounds was rapid loss through the kidneys.

Hyporesponsiveness to the immunosuppressant effects of delta-8-tetrahydrocannabinol.

Delta-9-tetrahydrocannabinol (delta 9-THC) and delta 8-THC have previously been shown to suppress humoral immunity in mice. The purpose of these investigations was to determine whether a hyporesponsiveness develops to the immunosuppressive effect of delta 8-THC. BALB/c mice were administered 60 mg/kg delta 8-THC i.p. 2 days after an i.p. immunizing dose of sheep erythrocytes (sRBC). Significant inhibition of direct hemolytic plaque-forming cells (PFC)/spleen was observed on days 3--6, with peak day of inhibition occurring on day 4. The effective inhibiting dose 50 (ED50) measured on peak day of response was 40 mg/kg for PFC/10(6) spleen cells and 38 mg/kg for PFC/spleen. When mice were pretreated daily for 5 days with different doses of delta 8-THC, similar ED50's were detected. Under this pretreatment regimen, 5 or 10 mg/kg produced no immunosuppression. Daily treatment with 5 or 10 mg/kg delta 8-THC for 5 days prior to sRBC and varying doses of delta 8-THC administered 2 days after sRBC resulted in significantly higher ED50's and increased values for the slopes of the dose response curves. These results suggest that a hyporesponsiveness develops to the immunosuppressive activity of delta 8-THC.