Extrapolation of experimental safety data to humans: the interleukin-6 case.

Interleukin 6 (IL-6) has a broad spectrum of biological activities, which include stimulation of hemopoiesis, especially thrombopoiesis, of the immune response, proliferation of mesangium cells, and induction of acute-phase proteins in the liver. Therefore, the therapeutic use of IL-6 for any specific clinical indication, e.g., stimulation of thrombopoiesis in severe thrombocytopenia, may be complicated by nonspecific effects. Preclinical experimental investigations of the safety evaluation performed in primates and rodents showed that rhIL-6 is well tolerated. A two- to three-fold increase in thrombocyte counts was observed along with an increase in acute-phase proteins and immunostimulation in the absence of target organ toxicity. Patients receiving rhIL-6, however, had a vigorous acute-phase response, with fever, anemia, and general malaise. Importantly, rhIL-6 administration did not cause mesangioproliferative nephritis or an uncontrolled lymphoproliferation as predicted from IL-6 transgenic mice. Although preclinical investigations are in general quite predictive for humans, safety data should be extrapolated carefully, since important quantitative differences may occur.

Recombinant human interleukin-6: safety issues of a pleiotropic growth factor.

Interleukin-6 (IL-6) plays a pivotal role in hematopoiesis, immune reactions and acute phase responses. It has been demonstrated, however, that the abnormal expression and dysregulation of IL-6 homeostasis are involved in the pathogenesis of a range of diseases. It is the purpose of this article to review briefly some of the biological properties of IL-6 in order to discuss issues related to the safety evaluation of recombinant human IL-6 (rhIL-6). Knowledge of the biological properties is important for, (1) selection of a responsive species for toxicological studies and (2) proper interpretation of the side-effect profile.

Dog neutrophil stimulation and cell surface expression of heat shock proteins induced by AHN 086.

The effect of AHN 086 [1-(2-isothiocyanatoethyl)-7-chloro-1,3-dihydro-5-(4- chloro-phenyl)-2H-1,4-benzodiazepine-2-one hydrochloride], an irreversible ligand of the mitochondrial benzodiazepine receptor, was studied in the heat shock protein (HSP) expression and the activation of dog neutrophils. At micromolar concentrations, AHN 086 induced a dose-dependent increase in free radical production (309 +/- 13% at 1 x 10(-5) M). This compound also increased the phagocytic activity (73 +/- 3.5% at 5 x 10(-6) M). Immunohistochemical results showed that AHN 086 induced the expression of HSP in neutrophil membranes. At 1 x 10(-5) M, the percentage of positive cells was: 90.2 (27 kD), 90.6 (72 kD) and 92.8 (90 kD). This expression was accompanied by a decrease in the presence of nuclear and cytoplasmic HSP. Flow cytometry studies showed that this cell membrane expression was located on the neutrophil surface and could be a consequence of HSP mobilization from nucleus and cytoplasm.

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.

Long-term interleukin-6 administration stimulates sustained thrombopoiesis and acute-phase protein synthesis in a small primate--the marmoset.

Interleukin-6 (IL-6) has been ascribed significant roles in both hematopoiesis and the immune response, although its contribution to host defence as a whole is poorly understood. Because short-term IL-6 treatment was previously shown to stimulate megakaryocytopoiesis, we investigated the effect of long-term administration of IL-6 on megakaryocytopoiesis and other systemic parameters in nonhuman primates. We chose a small primate, the marmoset (Callithrix jacchus), which enabled long-term administration at high doses. Recombinant human IL-6 (rhIL-6) administered at doses of up to 1,000 micrograms/kg/d over 4 and 9 weeks caused a sustained twofold to threefold increase of thrombocyte counts, peaking at 4 weeks. Thrombocyte counts declined thereafter, despite continuing IL-6 administration. The number of bone marrow megakaryocytes at 4 and 9 weeks was not increased compared with controls, but the ploidy grade was augmented, suggesting that IL-6 effects are restricted to mature megakaryocytes in vivo. An acute-phase protein response was observed within 24 hours after the first IL-6 administration and reached a maximum after 1 week of IL-6 administration at 25 micrograms/kg. Serum C-reactive protein, haptoglobin, and ceruloplasmin were increased, whereas albumin and transferrin levels declined. The acute-phase protein response was not associated with any morphologic evidence of hepatocellular damage. The increased levels of Ig and soluble IL-2 receptor in the serum levels reflected systemic immunostimulation. There was no evidence of renal mesangioproliferative pathology. Antibodies against rhIL-6 developed within 2 weeks, continuously increasing during the course of the study. High titers of neutralizing antibodies appeared concomitantly with the decrease in platelet counts and decline in acute-phase proteins. Therefore, despite the pleiotropic effects of IL-6 observed in vitro, long-term administration of IL-6 caused a selective and sustained stimulation of thrombopoiesis in marmosets that was only ablated by the appearance of neutralizing antibodies, and high doses were well tolerated in marmosets. A long-term targeting of IL-6 to cells of the megakaryocytic lineage, without evoking general toxicity, confirms the potential therapeutic usefulness of rhIL-6 for the chronic treatment of thrombocytopenic patients.

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.

Long-term treatment with 5,5-diphenylhydantoin reduces lymphadenopathy and anti-ssDNA autoantibodies in C57BL/6-lpr/lpr mice.

To further the insight in the immunomodulating properties of the anticonvulsant 5,5-diphenylhydantoin (DPH), C57BL/6 (B6), C57BL/6-lpr/lpr (B6-lpr/lpr) and MRL/MpJ- +/+ (MRL) mice received DPH orally for six months to determine weekly urinary biopterin levels, a potential T-cell activation marker, by high performance liquid chromatography. At the end of the experiment serum antibody levels were measured by ELISA and relative lymphoid organ weights determined. DPH treatment resulted in reduced body weight in all strains, reduced spleen weights in B6 and MRL mice, profoundly reduced popliteal lymph node weights in B6-lpr/lpr mice and increased thymus weights in MRL mice. DPH treatment decreased serum IgM, IgG and IgA as well as IgM and IgG anti-ssDNA levels in B6-lpr/lpr mice, but did not affect these parameters in other strains. Effects of DPH on IgM rheumatoid factor levels in B6-lpr/lpr mice were inconsistent. Urinary biopterin levels of untreated B6 and B6-lpr/lpr mice were about equal and lower than those of MRL mice. During the first three months of DPH treatment, persistently elevated biopterin levels were observed in B6 and to a lesser degree in MRL mice, and alternately elevated and control levels in B6-lpr/lpr mice. Thereafter, the effects faded in all strains. Results show that long-term DPH treatment causes only minor lymphoid organ weight changes in B6 and MRL mice, but causes a clear reduction of the lymphadenopathy and (auto)antibody formation in B6-lpr/lpr mice. Observed changes could not be related to altered biopterin excretion indicating that the latter is an inappropriate marker of murine autoimmune disease.

Cataractogenic effects in rats following chronic administration of SDZ ICT 322, a selective 5-HT3 antagonist.

SDZ ICT 322, an indole-3-carboxylic acid scopine ester developed for the treatment of delayed gastric emptying, was administered in feed to four groups of 14 male and 14 female Wistar rats at daily doses of 0, 5, 25 or 125 mg/kg/day for 26 weeks. Clinical and periodic ophthalmic examinations, hematology, clinical biochemistry, and urinalysis were performed. Eyes were examined for histopathologic and electronmicroscopic alterations as well as for reduced glutathione (GSH) and oxidized glutathione (GSSG) levels. Skin effects with hair loss, hyperemia, and desquamation were found in the majority of the high-dose rats. At study termination, bilateral posterior lens opacities had developed in 26 of the 28 high-dose rats. Histopathologically, grossly irregular and fragmented lens fibres were observed. Severe vacuolization of the lens epithelium was seen electronmicroscopically. The GSH/GSSG ratio was statistically significantly reduced in lenses of mid- and high-dose animals. Since decreased GSH/GSSG ratio is an established indicator for increased oxygen radical generation, these data suggest a possible role of oxidative stress in cataracts induced by SDZ ICT 322.

Pathology induced by interleukin-6.

Interleukin 6 (IL-6) is a multi-functional cytokine which plays an important role in the immune response, hemopoiesis and host defense. Recombinant human IL-6 (rhIL-6) was administered at high doses to mice, rats and non-human primates. In all species IL-6 had an immunostimulatory and hemopoietic (especially on megakaryocytes) effect. An acute phase response was most pronounced in non-human primates, which was not, however, associated with any major histopathological liver change. Finally, no evidence of glomerular pathology was found. Neutralising antibodies were detected within 10 days of rhIL-6 administration in all species.

V beta repertoire in rats and implications for endogenous superantigens.

Endogenous superantigens of mice, encoded by mammary tumor virus proviral integrants, induce intrathymic deletion of entire T cell populations that express specific V beta gene products, a phenomenon proposed to be important in self-tolerance and prevention of toxic responses to exogenous microbial superantigens. Evidence for the presence of V beta selecting/deleting endogenous superantigens in other species is lacking. We report here that rats do not exhibit endogenous superantigen-induced V beta clonal deletions despite their strong response to bacterial superantigens. These findings indicate that endogenous superantigens are not obligatory in V beta repertoire shaping.

Urinary biopterin levels in mice during graft-versus-host reactions and during exposure to 5,5-diphenylhydantoin.

Based on evidence that urinary neopterin levels are useful markers of disordered cellular immunity in man, we investigated murine urinary biopterin excretion during acute and chronic graft-versus-host (GvH)-reactions as well as after oral exposure to drugs with documented immune disregulating potential in man. Biopterin levels were determined in urine spot samples by reversed-phase high performance liquid chromatography and expressed in relation to the urinary creatinine content. Similarly increased and decreased biopterin levels were observed during acute and chronic GvH-disease in (C57BL/6J x DBA/2J)F1 (B6D2F1) mice. Increased and/or decreased levels of urinary biopterin were observed during treatment with 5,5-diphenylhydantoin (DPH), methimazole, propylthiouracil and nitrofurantoin, but no consistent pattern could be distinguished. The DPH-induced alterations were similar in B6 and B6D2F1 mice, were dose-dependent, reversible and independent of mature T-cells, as judged by the pronounced biopterin excretion of B6-nu/nu mice in comparison with their T-cell competent litter mates. The results indicate that monitoring of urinary biopterin excretion in mice does not represent a useful biochemical marker for T-cell activation.

Kinetics and morphology of chemically induced popliteal lymph node reactions compared with antigen-, mitogen-, and graft-versus-host-reaction-induced responses.

Changes in the popliteal lymph node (PLN) in mice evoked by a local graft-versus-host (GVH) reaction and by a single injection of various agents into the hind footpad were compared. The drug diphenylhydantoin induced similar weight changes in time as the GVH reaction. More vigorous and protracted reactions were induced by the drug nitrofurantoin and the contact sensitizer dinitrochlorobenzene, whereas the antigens lipopolysaccharide and sheep erythrocytes caused very moderate and short-lasting weight changes. Alterations of lymph node architecture upon injection of diphenylhydantoin resembled those observed during the GVH response. Some quantitative and qualitative differences were noted for nitrofurantoin, but clearly deviant morphological alterations were seen in response to lipopolysaccharide and sheep erythrocytes. The PLN reaction to dinitrochlorobenzene had features of both the GVH reaction and the antigen-induced responses. These findings support the concept that some drugs and chemicals may induce or exacerbate lymphoproliferative disorders by GVH-like mechanisms.

The popliteal lymph node assay in mice to screen for the immune disregulating potential of chemicals--a preliminary study.

Low mol. wt compounds were tested in the popliteal lymph node (PLN) assay to study whether PLN reactivity could be related to the ability of the compounds to induce autoimmune disorders in man. PLN reactions were measured 7 days after a single subcutaneous (s.c.) injection of dissolved compounds in amounts of 0.3-2.0 mg into one hind footpad of mice and assessed as the weight increase of the draining PLN relative to the PLN weight of the untreated contralateral paw. Hydralazine, chlorpromazine, diphenylhydantoin, carbamazepine, phenylbutazone and nitrofurantoin, all being drugs with a documented potential to induce systemic immunological disorders in man, caused marked PLN reactions. False negative PLN responses were observed following injection of procainamide and isoniazid. Among systemic drugs without known potential to induce autoimmune reactions in humans, quinacrine, denzimol and niridazole significantly increased PLN weights, while phenobarbital, levamisole and disulfiram had no effect. Chemicals with a well-known capacity to induce contact dermatitis in man like 2,4-dinitro-1-chlorobenzene, alpha-methylene-gamma-butyrolactone, p-phenylenediamine, 5-nitro-2-furaldehyde semicarbazone, 2-mercapto-benzothiazol and 1,3-dibutyl-2-thiourea caused marked PLN reactions, while the non-sensitizer 2,4-dichloro-1-nitrobenzene failed to do so. It is concluded that the PLN assay as applied in this study may give a rapid first indication of immunomodulating potential of low mol. wt compounds, but it does not discriminate as to the kind of immunomodulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Popliteal lymph node reactions in mice induced by the drug zimeldine.

The antidepressant drug zimeldine was screened for immune modulating properties using the popliteal lymph node (PLN) assay as a test system in mice. In immunocompetent as well as congenitally athymic nude mice, footpad injection of 1.0 mg zimeldine triggered a bimodal footswelling. A transient oedematous swelling, histologically characterized by mast cell degranulation, was followed by infiltration of polymorphnuclear cells. A dose-dependent PLN enlargement to the agent was observed, which appeared to be more pronounced in immunocompetent mice as compared with athymic nude mice, and in H-2b mice as compared with H-2d mice. After injection of 1.0 mg zimeldine into the footpad of C57BL/10 mice, significant enlargement was already observed by 3 days after injection, was optimal around day 9 and persisted for at least 30 days. Histologically, PLN reactions were characterized by blast transformation of lymphocytes and expansion of paracortical areas prior to germinal center reactions in enlarged follicles. Size of both areas gradually decreased as the medulla filled with plasma cells, 7-30 days after injection. The observed reactions could not be transferred with syngeneic lymph node cells after prior exposition to zimeldine in vivo or in vitro. We conclude that zimeldine induces strong and persistent PLN enlargement, blastogenesis and prominent germinal center reactions. Immunocompetent T-cells are apparently conducive, but not prerequisite to these reactions, which suggests involvement of multiple mechanisms including those mediated by inflammatory reactions in the foot. It is unlikely that the observed enlargement of PLN can be attributed to a direct chemical modification of leukocyte membranes by zimeldine. The protracted nature of the reaction may indicate that zimeldine somehow interferes with inhibitory feedback mechanisms.

1-Phenyl-5-vinyl-2-imidazolidinethione, a proposed causative agent of Spanish toxic oil syndrome: synthesis, and identification in one of a group of case-associated oil samples.

A method is described for the synthesis and characterization of N-(2-hydroxy-3-butenyl)-N'-phenylthiourea, and its cyclization product, 1-phenyl-5-vinyl-2-imidazolidinethione (PVIZT). Fourteen coded oil samples associated with toxic oil syndrome cases in Spain were examined by gas chromatography-electron impact mass spectrometry for the presence of PVIZT. Although these samples were obtained from households where cases of toxic oil syndrome had been recorded, they differed extremely with regard to their anilide and sulphur contents. In one sample PVIZT was detected at an estimated concentration of 1 mg/kg.

Structural requirements for hydantoins and 2-thiohydantoins to induce lymphoproliferative popliteal lymph node reactions in the mouse.

The ability of a large number of hydantoins and 2-thiohydantoins to induce primary local lymphoproliferative popliteal lymph node (PLN) reactions has been investigated, as judged by PLN weight enlargement, in an attempt to evaluate the discriminating potential of the PLN reaction to low mol. wt chemicals and to establish structure-activity relationships. Among a series of nineteen hydantoins and related compounds only 5,5-diphenylhydantoin (phenytoin), its major metabolite 5-(p-hydroxyphenyl)-5-phenylhydantoin, 5,5-diphenyl-2-thiohydantoin and N-(5-nitro-2-furfurylidene)-1-aminohydantoin (nitro-furantoin) elicited marked PLN reactions in C57BL/6J mice. In DBA/2 mice, PLN responses to the aforementioned compounds were considerably less or virtually absent. A number of hydantoin derivatives and related compounds with one phenyl group and/or other substituents at the 1,3 or 5 position induced only slightly elevated or suppressed PLN responses in C57BL/6J mice. The influence of polar, and lipophilic aliphatic and aromatic substituents at the 5 position were compared among a series of 22 3-methyl-2-thiohydantoin as well as 21 3-phenyl-2-thiohydantoin amino acid derivatives for their ability to elicit primary PLN reactions in C57BL/6J mice. Substitution with only one aromatic group at the 5 position seemed to be necessary to induce PLN enlargements to 2-thiohydantoins already substituted at the 3 position with a methyl group or even more pronounced when substituted with a phenyl group. p-Hydroxylation of 5-benzyl-3-phenyl-2-thiohydantoin significantly diminished the PLN response. In contrast, p-hydroxylation of one of two phenyl groups as in 5-(p-hydroxyphenyl)-5-phenylhydantoin had little effect on lymphoproliferative PLN reactions. The presence of a hydroxyl group in a non-aromatic cyclic substituent as in hexahydro-6-hydroxy-2-methyl-3-thioxo-1H-pyrrolo[1,2-c]imidazol-1- one had no effect on the PLN reaction. A series of aliphatic substituents in the 5 position of 2-thiohydantoins showed that the number of carbon atoms of the substituents as well as the position of side chains in the isomer, rather than the methyl or phenyl group in the 3 position of the 2-thiohydantoin molecule, determined the strength of the PLN enlargement. It is concluded that the PLN weight increase assay appears to be able to discriminate between subtle chemical differences as studied with a large series of hydantoin and 2-thiohydantoin derivatives. The PLN assay may therefore be useful as a preliminary short-term screening method for identification of (classes of) compounds able to induce lymphoproliferative reactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Chemical-induced autoimmune reactions and Spanish toxic oil syndrome. Focus on hydantoins and related compounds.

Autoimmune diseases comprise a wide spectrum of overlapping, systemic and organ-specific disorders. Although, etiology and pathogenesis of such disorders are largely unknown, endogenous host factors and exogenous agents, such as viruses, bacteria, and small molecular weight chemicals, drugs and food components, are believed to be involved. The toxicological significance of low molecular weight compounds on induction of autoimmune disorders is illustrated by the toxic oil syndrome (TOS), a chemically induced epidemic, observed in Spain since 1981. The causative chemical(s) of TOS is still elusive, but an association between ingestion of refined aniline-adulterated rapeseed oil and the syndrome is well-documented. Epidemiological, clinical and immunopathological symptoms of TOS are briefly reviewed. The striking resemblance with immunological disorders, observed in man upon medication with hydantoins and related compounds, is demonstrated. The likeliness of formation of a hydantoin-related compound in the aniline-adulterated oil is evidenced and its role as possible toxic agent in TOS is proposed. Further, the presence of hydantoins and related compounds in food is briefly reviewed and it is suggested that these chemicals may account for a portion of idiopathic autoimmune diseases observed in man. The need for development of animal models to assess this kind of immunotoxicological effects is stressed.