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This chapter describes the use of ultrathin oxide overlayers to modify and control the properties of electrocatalysts. Of particular interest are oxide-encapsulated electrocatalysts (OECs), for which the oxide overlayers are permeable to electroactive species such that electrochemical reactions occur at the buried interface between the overlayer and active electrocatalyst component. Such OECs offer several advantages over conventional electrocatalysts that are directly exposed to the electrolyte, presenting opportunities to improve the durability, selectivity, and activity of the active electrocatalyst material. However, the intertwined nature of the physical and chemical phenomena that underlie OEC operation adds complexity to the task of rationally designing OECs, and an improper choice of operating conditions or overlayer characteristics can be highly detrimental to their performance. To help researchers overcome these challenges, this chapter describes (i) the transport and kinetic principles that underlie OEC operation and (ii) experimental tools and methodologies that can be used to evaluate OEC performance. Collectively, the principles and methodologies described here can serve as a framework for developing design rules for optimized OECs that can be applied to a wide range of electrochemical energy applications.

Salts of 1,2,4-triazole and 3-bromo-1,2,4-triazole with Cd, Zn, Co, Ni, Cu and Ag are reported. The compounds have been identified and characterized by chemical analyses, vibrational and electronic absorption spectra. Different coordination possibilities are discussed in view of the observed vibrational spectra. For the salts of 1,2,4-triazole, 1,2-bidentate or tridentate coordinations are observed. In the salts of 3-bromo-1,2,4-triazole, bidentate and tridentate coordinations are assumed.

Atomic force microscopy and synchrotron radiation (SR) spectroscopy have been used to study the wetting behavior and electronic structure of thin films of a novel organometallic cluster--[BuSn(O)OC(O)Fc]6 ("Fc" = ferrocenyl)--on silicon substrates. This cluster comprises six ferrocene units connected to a stannoxane central core--"an iron wheel on a tin drum" (V. Chandrasekhar; et al. Angew. Chem., Int. Ed. 2000, 39, 1833). Thin films spin-cast onto native oxide-terminated silicon readily dewet the substrate. We have utilized advanced image analysis techniques based on Minkowski functionals to provide a detailed quantitative analysis of the morphology of the stannoxane overlayers. This analysis shows that the dewetting patterns are rather far removed from those expected to arise from a simple Poisson distribution of centers, and we discuss the implications of this finding in terms of nucleated and spinodal dewetting. Variations in both the surface roughness and the in-plane correlation length have been followed as a function of annealing time to probe the surface dewetting dynamics. SR valence band photoemission illustrates that the highest occupied molecular orbital (HOMO) of the cluster is found 2 eV below the Fermi level. Fe 2p --3d and Sn 3d --5p resonant photoemission spectroscopy have been used to enhance the cross sections of the partial density of states associated with the Fe and Sn atoms. Sn atoms make a large contribution to the HOMO of the cluster, whereas the Fe atoms are associated with an electronic environment seemingly very similar to that in the "parent" ferrocene molecule.

The use of scanning electrochemical microscopy (SECM) to measure charge transport diffusion constants (DCT) in metallopolymers of the type [Os(bpy)2(PVP)nCl]Cl, bpy = 2,2‘-bipyridyl and PVP = poly(4-vinylpyridine), is described. In this approach, a triple potential step technique is employed in which the ultramicroelectrode (UME) tip of the SECM is used to electrogenerate a solution phase oxidant, Ru(CN)63-, in an initial potential step, via the oxidation of Ru(CN)64-. This moiety diffuses from the tip to the underlying polymer film where electron transfer occurs, causing the local oxidation of the polymer-bound complex of OsII to OsIII. The form of the current−time characteristic in this step provides information on the kinetics of the ET process between the solution species and the polymer-bound moiety, as well as the concentration of redox-active species in the polymer film. This process creates lateral concentration gradients of OsII and OsIII along the film. After the first potential step, a waiting ...

The threshold of toxicological concern (TTC) is a pragmatic risk assessment tool that is based on the principle of establishing a human exposure threshold value for all chemicals, below which there is a very low probability of an appreciable risk to human health. The concept that there are levels of exposure that do not cause adverse effects is inherent in setting acceptable daily intakes (ADIs) for chemicals with known toxicological profiles. The TTC principle extends this concept by proposing that a de minimis value can be identified for many chemicals, in the absence of a full toxicity database, based on their chemical structures and the known toxicity of chemicals which share similar structural characteristics. The establishment and application of widely accepted TTC values would benefit consumers, industry and regulators. By avoiding unnecessary toxicity testing and safety evaluations when human intakes are below such a threshold, application of the TTC approach would focus limited resources of time, cost, animal use and expertise on the testing and evaluation of substances with the greatest potential to pose risks to human health and thereby contribute to a reduction in the use of animals. An Expert Group of the European branch of the International Life Sciences Institute-ILSI Europe-has examined the TTC principle for its wider applicability in food safety evaluation. The Expert Group examined metabolism and accumulation, structural alerts, endocrine disrupting chemicals and specific endpoints, such as neurotoxicity, teratogenicity, developmental toxicity, allergenicity and immunotoxicity, and determined whether such properties or endpoints had to be taken into consideration specifically in a step-wise approach. The Expert Group concluded that the TTC principle can be applied for low concentrations in food of chemicals that lack toxicity data, provided that there is a sound intake estimate. The use of a decision tree to apply the TTC principle is proposed, and this paper describes the step-wise process in detail. Proteins, heavy metals and polyhalogenated-dibenzodioxins and related compounds were excluded from this approach. When assessing a chemical, a review of prior knowledge and context of use should always precede the use of the TTC decision tree. The initial step is the identification and evaluation of possible genotoxic and/or high potency carcinogens. Following this step, non-genotoxic substances are evaluated in a sequence of steps related to the concerns that would be associated with increasing intakes. For organophosphates a TTC of 18microg per person per day (0.3 microg/kg bw/day) is proposed, and when the compound is not an OP, the TTC values for the Cramer structural classes III, II and I, with their respective TTC levels (e.g. 1800, 540 and 90 microg per person per day; or 30, 9 and 1.5 microg/kg bw /day), would be applied sequentially. All other endpoints or properties were shown to have a distribution of no observed effect levels (NOELs) similar to the distribution of NOELs for general toxicity endpoints in Cramer classes I, II and III. The document was discussed with a wider audience during a workshop held in March 2003 (see list of workshop participants).

Hyperplastic and metaplastic lesions in the prostate gland of bulls are used as morphological evidence for the illegal administration of oestrogens or compounds with oestrogenic activity, like stilbenes. Exogenous and endogenous androgens can suppress the effects of these drugs, resulting in suspect or negative results. The detection of epithelial changes immunohistochemically with cytokeratin antibodies appeared to facilitate the diagnosis of hyperplasia and metaplasia in the prostate. A specific and sensitive method for detecting such lesions in formalin-fixed, paraffin-embedded prostate samples has been developed with the commercially available cytokeratin monoclonal antibody 34 beta E12, and applied to bulls treated with diethylstilbestrol-dipropionate. The highly reproducible staining pattern of 34 beta E12 can be used for the detection of hyperplasia and metaplasia in the prostate glands of animals treated illegally with oestrogens.

This paper presents an overview of the studies carried out in small laboratory fish species to investigate the usefulness of histopathology as a tool in aquatic toxicology. The studies were performed with medaka (Oryzias latipes) and guppy (Poecilia reticulata) that were exposed to a variety of environmental contaminants for one and three months. Besides the measurement of routine toxicological parameters, total body histopathology was carried out. The data are summarized and evaluated in view of their contribution to the knowledge of toxicology, the specific responsiveness of animals and tissues, and the application in ecotoxicology. It is concluded that histopathology of (small) fish exposed to environmental contaminants may provide useful information as to target organs and mechanism of action: moreover, this technique can be more sensitive than routine parameters.

Circular double-stranded replication intermediates were identified in low-molecular-weight DNA of cells of the avian leukemia virus-induced lymphoblastoid cell line 1104-X-5 infected with chicken anemia virus (CAV). To characterize the genome of CAV, we cloned linearized CAV DNA into the vector pIC20H. Transfection of the circularized cloned insert into chicken cell lines caused a cytopathogenic effect, which was arrested when a chicken serum with neutralizing antibodies directed against CAV was added. Chickens inoculated at 1 day of age with CAV collected from cell lines transfected with cloned CAV DNA developed clinical signs of CAV. The 2,319-bp cloned CAV DNA contained all the genetic information needed for the complete replication cycle of CAV. The CAV DNA sequence has three partially overlapping major reading frames coding for putative peptides of 51.6, 24.0, and 13.6 kDa. The CAV genome probably contains only one promoter region and only one poly(A) addition signal. Southern blot analysis using oligomers derived from the CAV DNA sequence showed that infected cells contained double- and single-stranded CAV DNAs, whereas purified virus contained only the minus strand. It is the first time that the genome of one of the three known single-stranded circular DNA viruses has been completely analyzed.

Aberrant or defective immune responses are known to be the result of primary immunodeficiency diseases, e.g., in the DiGeorge syndrome (thymic hypoplasia) in man, or in homozygous mice with the mut...

In the 2:1 co-crystal of 5-(2-pyridyl)-1H-1,2,4-triazole (pyt) and 1,4-dihydroxybenzene (dhb), 2C 7 H 6 N 4 -C 6 H 6 O 2 , the pyt molecules form two independent N-H...N hydrogen-bonded chains. The dhb molecules act as hydrogen-bonding bridges between the two pyt chains via two O-H...N hydrogen bonds. The complex is further stabilized by a number of weaker interactions, including C-H...N, C-H...O and C-H...π interactions.

This paper briefly reviews the application of histopathology as aninstrument or endpoint in toxicity studies in fish. For long this has been applied rather occasionally in (regulatory) toxicology, and was mainly of interest in fundamental studies and limited carcinogenicity experiments. However, nowadays there are various incentives that ask for the application of pathology, such as field monitoring of pollution effects, the wish for optimal use and lower species of laboratory animals, the availability of modern histology techniques, and insight and interest in mechanistic data. This is timely illustrated by the current broad interest in endocrine disrupting pollutants-a threat mainly in the aquatic environment-where histopathological organ and tissue changes in intact sentinel fish species provide pivotal diagnostic and mechanistic features.

In recent years, mass mortalities among seals and dolphins have been attributed to infections with different morbilliviruses. In all cases, these marine top predators were exposed to high levels of persistent lipophilic environmental contaminants accumulated through the food chain. This observation led to the hypothesis that a contaminant-related suppression of the immune system might have contributed to the severity of the virus outbreaks. We conducted a semi-field feeding experiment, in which we fed two groups of harbour seals (Phoca vitulina) fish with different levels of contaminants. During a period of 2 1/2 years, blood samples were taken at regular intervals, and the functioning of different compartments of the immune system was monitored and compared. We found impaired natural killer (NK) and specific T cell responses in the seals fed contaminated fish. This is the first demonstration of immunosuppression in mammals following chronic exposure to environmental contaminants at ambient environmental levels.

Sublethal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces thymic atrophy and thymic lymphodepletion in all species investigated. Lymphodepletion is first seen in the cortex of the thymus, but also occurs in the medulla at later time points. This article surveys in vivo and in vitro studies on TCDD-induced thymus toxicity. It is concluded that the first intrathymic target cell for the action of TCDD is the cortical epithelial cell.

The charge transport behaviour of glassy-carbon electrodes modified with the osmium containing polymer [Os(bipy)2(PVI)nCl]Cl, where bipy = 2,2’ — bipyridyl, PVI is poly — N vinylimidazole and n = 5, 10, 15, 20, 25 is reported. The polymer coatings behave like ideal surface bound redox couples and standard theoretical models have been used to evaluate the charge transport process. The charge transport parameters are found to be strongly dependent on the nature and concentration of the electrolyte. The effect of the loading of the redox centre (n) is also considered. The diffusion coefficients obtained depend on the time domain used for the measurement Activation parameters have been obtained to probe the nature of the rate-limiting step in the charge-transport process. The results are compared with those obtained for the analogous polymers [Os(bipy)2(PVP)nCl]Cl, where PVP = poly4-vinylpyridine.

We studied the effect of Cyclosporin A on the thymus in rats. A daily subcutaneous injection of Cyclosporin A at a dose of 45 mg/kg body weight in young adult rats resulted in a 100% mortality within two weeks. Daily administration of 15 mg/kg for two weeks was well tolerated. Rats treated at this dose showed whole blood Cyclosporin concentrations around 6 mg/l. The thymus immediately after treatment showed the almost complete disappearance of the medulla, including the microenvironment made by medulla-type epithelium, interdigitating dendritic cells defined by their expression of MHC class II antigen, and lymphocytes with a mature T-cell phenotype. After discontinuation of Cyclosporin treatment, a rapid recovery occurred, with the reappearance of medulla areas after 2 weeks. These areas differed from the normal medulla by the absence of medulla-type epithelium. This cell population recovered in its normal location in about 4 weeks. The reappearance of medullary interdigitating cells was associated with reappearance of lymphocytes with a mature T-cell phenotype. In the regenerating thymus "holes" in the microenvironment were observed that lacked epithelium and interdigitating cells, and that were filled by lymphocytes with an immature cortex phenotype. Peripheral lymphoid organs, of which the spleen was studied in more detail, did not manifest changes in lymphoid and stromal components. Target organs for syngeneic graft-versus-host disease, as described under special conditions after Cyclosporin treatment, did not show any histologic abnormality. The changes in thymus (immuno)histology may be associated with changes in shaping the T-cell repertoire: but, clinical manifestations of such changes require special experimental conditions.

In this experiment 20 one year old bulls received a single intramuscular injection of the anabolic preparation diethylstilbestrol dipropionate (DES-DP) (an oil preparation or an emulsion). Four animals received a corresponding placebo. The application of DES-DP to bulls caused characteristic histological alterations in the peripheral glandular epithelium of the prostate, which could be observed until four weeks after treatment. The value of histological investigation as a screening method was, however, limited by the occurrence of only few metaplastic lesions and a rapid recovery. By contrast, immunohistochemistry using a polyclonal cytokeratin antiserum K40 appeared to be a specific and very sensitive method to detect oestrogen-induced lesions in the prostate. In only two animals, six weeks after injection with the DES-emulsion, false-negative results were obtained, demonstrating the potential value of this screening method. The excretion of DES in the urine and faeces was monitored using radioimmunoassay following chromatographic purification of the urine and faeces extracts. The excretion of DES in urine was faster for animals of the oil group. The DES content in urine decreased to the 1 microgram/l level after 42 days (emulsion group) or 70 days (oil group). The excretion in faeces was comparable to that in urine. After day 21 the excretion patterns of the two excreta were indistinguishable.

Rats were fed diets containing 0, 500, 1000, and 2000 mg HCB/kg during a 3-week period. Marked weight increases of spleen, popliteal and mesenteric lymph nodes and of the liver were found. Histologically, the white pulp in the spleen was enlarged because of an increase in size of marginal zones and follicles. In addition, there was an increase of extramedullary hemopoiesis. In the lymph nodes, the number of high endothelial venules was increased at all dose levels. The number of neutrophils, basophils and monocytes in the peripheral blood was significantly increased, whereas peripheral lymphocyte counts were slightly higher. Total serum IgM levels were markedly increased, but IgG concentrations were unaltered. On the basis of this experiment, the 1000 mg HCB/kg diet level was chosen for the different function studies that were carried out after a 3-weeks dietary regimen. Regarding the humoral immunity, IgM antibodies to LPS were unaltered, whereas primary and secondary IgM and IgG antibody titers to tetanus toxoid were increased approximately three-fold. HCB did not significantly alter the cell-mediated immunity, as shown by the following parameters: resistance to Listeria monocytogenes infection, rejection of skin transplants, and delayed-type hypersensitivity to tuberculin. The phagocytizing capacity of macrophages was studied by measuring the blood clearance of carbon particles. HCB did slightly depress the phagocytic index, but the difference with control animals was statistically not significant. The in vitro responsiveness of thymus cells to the mitogens PHA, Con A, and PWM was not changed by in vivo HCB-treatment. On a cell-for-cell basis, the responsiveness of spleen cells was increased when cultured in the presence of LPS. On a whole organ basis, the response to PHA, Con A, PWM, and LPS was markedly enhanced because of an increase in the number of nucleated spleen cells. Regarding peripheral lymphocytes, only the response to the mitogen Con A was higher. On the basis of these studies it is concluded that HCB stimulates the humoral immune response in the rat, enhances the in vitro responsiveness of spleen cells to the different mitogens mainly as a result of an increase in the number of splenic lymphocytes, but does not alter the cell-mediated immunity as shown with in vivo tests. This result contrasts with data in the literature that show that HCB suppresses the humoral and cell-mediated immunity in mice. Finally, HCB pretreatment only marginally increased the susceptibility of rats to endotoxin, whereas mice have been shown to be 20-fold more susceptible to the lethal effects of bacterial endotoxin.

Three-month-old male C57B1 6 mice were given single oral doses of 0, 100, 150, or 200 μg of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg. The LD50 was 114 μ/kgg. In mice that died, depletion of the thymus and spleen were consistently found and edema and terminal hemorrhages occurred frequently. In a second experiment, 4-month-old male mice were dosed po with 0, 0.2, 1.0, 5.0 or 25 μg/kg, once a week for 2 or 6 weeks. Some deaths and growth retardation occurred in the 25 μg/kg dose group. Significantly increased liver and decreased thymus weights were found in the 1, 5 and 25 μg/kg dose groups. Total neutrophils were increased significantly, whereas hemoglobin values and mean corpuscular hemoglobin concentrations were decreased significantly after 6 doses of 25 μg/kg. Total serum protein and α-, β-, and γ-globulins were significantly decreased. TCDD was porphyrogenic. The hepatic porphyria was probably associated with liver damage. Degenerative and necrotic changes in the liver were essentially centrilobular and were accompanied by cellular infiltrates and ceroid pigment deposition. Proliferation of bile duct and bile duct epithelial cells occurred. Lipid accumulation was centrilobulary localized in the mice receiving 0.2 μg/kg, was more pronounced in the mice of the intermediate dose levels, and involved hepatocytes throughout the lobule in the 25 μg/kg dose group.

Based on an earlier described macromethod for the routine measurement of IgM and IgG in rat sera, a mechanized micro enzyme-linked immunosorbent assay (ELISA) was developed. The assay was performed in the wells of microtiter plate thus minimizing the quantities of reagents and antisera needed. Data on reproducibility of the assay and calculation of IgM and IgG levels are provided. For the functional assessment of the humoral immunity of the rat, ELISA is a powerful tool. In an earlier report, assays for the titration of thymus-independent IgM antibodies to E. coli LPS and the IgM and IgG response to the thymus-dependent antigen tetanus toxoid were described. More recently it was shown that the antigen ovalbumin elicits a thymus-dependent IgM, IgG and IgE response which could be readily measured with the enzyme immunoassay, as well as a delayed-type hypersensitivity reaction. As the optimum ovalbumin concentration for both types of reactions was the same, it is concluded that the ovalbumin model offers the advantage that both humoral and cellular immunity can be studies simultaneously in the same animal. Images FIGURE 1.

Rat IgM and IgG was determined by mechanized "sandwich" enzyme-linked immunosorbent assay (ELISA) using peroxidase labeled anti-rat-IgM and -IgG. Linear ranges in standard curves of a reference rat serum had a slope similar to the slopes found with sera of 25 rats of various age. IgM and IgG measurements by ELISA in these sera correlated well with results obtained by single radial immuno-diffusion (SRID). In addition, the precision of the enzyme immunoassay was the same as obtained with the SRID. Compared with SRID, ELISA is less time consuming and the amount of antiserum used in the macro-ELISA is one order of magnitude lower; and again 10 times lower in the mechanized micro-ELISA that is currently being developed. In conclusion, the ELISA is a specific, reliable, sensitive, and economic method for routine measurement of rat serum IgG and IgM e.g. in toxicity studies. In the second part of this study, ELISA and the passive hemagglutination test were compared to determine the primary and secondary antibody response to E. coli lipopolysaccharide (LPS) and tetanus toxoid in rats. In the ELISA, the antigens were bound to the wells of polystyrene microplates. Tetanus toxoid was coated directly, LPS after complexing with methylated bovine serum albumin. After incubation with dilutions of the rat sera, the amount of antibody bound to the solid phase was quantified by means of peroxidase-labeled antiimmunoglobulin. The specificity of the enzyme immunoassay was tested by absorption of the sera with the respective antigens. ELISA proved to be more sensitive than the hemagglution reaction, except when titers were determined during the secondary response to tetanus toxoid. Besides its specificity and sensitivity, ELISA is a convenient method for measuring both IgM and IgG antibodies. Finally, evidence is presented that in the rat, the humoral immune response to LPS is a thymus-independent phenomenon. Thus, by using the antibody response to LPS and tetanus toxoid in function studies of the immune system of the rat, insight can be obtained in the thymus-independent and thymus-dependent humoral immune response.

We studied the development of thymus-dependent immunity in congenitally athymic nude rats after implantation of cultured thymic fragments (CTF), particularly the development of in vitro alloreactivity in allogeneic combinations. CTF of DA (RT1a), PVG (RT1c), and RP (RT1p(u,1] origin were implanted in nude rats of WAG (RT1u) origin. In analysis 14 to 18 wk later, all recipients exhibited thymus-dependent immunocompetence assessed by (immuno)-histology of lymphoid organs and responsiveness to in vitro concanavalin A stimulation and in vivo ovalbumin immunization. Control nude animals were unresponsive. Also, in vitro alloreactivity was observed, measured by mixed lymphocyte reaction and cell-mediated lympholysis. The alloresponse to the allogeneic CTF donor haplotype was as to a third party, but that to the recipient was negative. The CTF before implantation were devoid of lymphoid elements and revealed epithelial-like cells as the major component. Cells in CTF showed expression of RT1 class I and class II antigens. CTF at autopsy had the architecture of a normal thymus. In immunohistochemistry using haplotype-specific antibodies, lymphocytes showed RT1u class I expression as in the normal WAG thymus. In the cortex-like area of CTF, stromal cells revealed class I and class II haplotype expression of the donor thymus, but in the medulla-like area, class II haplotype expression was that of the recipient WAG rat. These data indicate that after implantation in nude rats, CTF become populated not only with lymphoid elements, but also with stromal components from the recipient. In induction of thymus-dependent immunity, these acceptor-derived stromal (dendritic) cells may be involved in generation of allospecificity; class I and class II haplotype expression by the donor cortex (epithelial) compartment is ignored in this process.

Chlorodibenzo-p-dioxins, especially 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), are among the most toxic compounds known. These compounds are found as contaminants of technical chlorophenols and their derivatives. A variety of pathologic condition have been associated with the ingestion of products containing chlorodibenzo-p-dioxins. Toxic fat, the cause of chick edema disease (1, 2) and a variety of pathologic manifestations in monkeys (3), has been found to contain chlor3dibenzo-p-dioxins, among which is TCDD (4, 5). TCDD has been implicated in a variety of other toxicoses, including outbreaks of chloracne in chemical workers (6), hepatonecrosis in rabbits (6), and hepatonecrosis and thymic atrophy in rats (7). However, only in the studies in which monkeys were fed toxic fat (3) and in which rats were given TCDD orally (7, 8) were substantial clinical pathologic analyses performed. Toxic fat caused in the monkeys, among other lesions, anemia, leukopenia and hypoproteinemia. The hypoproteinemia was primarily due to decreased serum albumin concentration. The blood urea nitrogen

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is reported to be one of the most toxic chemicals known. There has been variation in such reported toxic parameters as single oral LD50 dose, range of time interval from dosing until death, and toxic manifestations which an animal exhibits (1-4). It seemed most appropriate, therefore, that the mean values and ranges of these and other general biological parameters be determined under the experimental condition to which animals are exposed at this Institute.

Images FIGURE 1. FIGURE 2. FIGURE 3. FIGURE 4. FIGURE 5. FIGURE 6. FIGURE 7. FIGURE 8. FIGURE 9. FIGURE 10. FIGURE 11. FIGURE 12. FIGURE 13. A FIGURE 13. B

Images FIGURE 1. FIGURE 2.

Images FIGURE 1. FIGURE 2. FIGURE 3. a FIGURE 3. b FIGURE 3. c FIGURE 3. d FIGURE 4. FIGURE 5 FIGURE 5 FIGURE 6. FIGURE 7. a FIGURE 7. b

As a screening procedure to detect immunotoxic effects, different parameters are discussed. These criteria include growth, weight and histology of lymphoid and endocrine organs, haematology, as well s serum immunoglobulin concentrations which can be quantified using the enzyme-linked immunosorbent assay (ELISA). Secondly, different function tests are discussed to assess in the rat the cell-mediated immunity, the humoral immunity and the phagocytosis by macrophages. These tests should be performed when in a screening study an indication of immunotoxicity is found at a relevant dose level. The aim of functional assessment is to determine the functional significance of an effect on the immune response and to gain more insight into the mode of action of the chemical. Finally, examples are given of compounds that suppress or stimulate immune responses; and advantages are discussed of performing function tests during the development phase of the immune system.

Antitumor activity, cardiotoxicity, and nephrotoxicity induced by doxorubicin were studied in LOU/M/WSL inbred rats each bearing a transplantable solid IgM immunocytoma. Animals with a tumor (diameter, 15.8 +/- 3.3 mm) were treated with iv injections of doxorubicin on 5 consecutive days, followed by 1 weekly injection for 7 weeks (dose range, 0.015-4.0 mg/kg body wt). Tumor regression was observed with 0.5 mg doxorubicin/kg. Complete disappearance of the tumor was induced with 1.0 mg doxorubicin/kg. Histologic evidence of cardiotoxicity scored as grade III was only observed at a dose of 1.0 mg doxorubicin/kg. Light microscopic evidence of renal damage was seen above a dose of 0.5 mg doxorubicin/kg, which resulted in albuminuria and very low serum albumin levels. In the group that received 1.0 mg doxorubicin/kg, the serum albumin level decreased from 33.6 +/- 4.1 to 1.5 +/- 0.5 g/liter. Ascites and hydrothorax were observed simultaneously. The same experiments were performed with non-tumor-bearing rats, in which no major differences were observed. In conclusion, antitumor activity, cardiotoxicity, and nephrotoxicity were studied simultaneously in the same LOU/M/WSL rat. Albuminuria due to renal damage led to extremely low serum albumin levels, so ascites and hydrothorax were not necessarily a consequence of the observed cardiomyopathy.

In a previous study on doxorubicin-induced cardiotoxicity in LOU/M/Wsl rats, severe nephropathy has been observed; therefore, the question was raised whether nephropathy adds to or even might be responsible for doxorubicin-induced cardiomyopathy in rats. For elucidation of this question, the temporal relationship between the onset of doxorubicin-induced cardiomyopathy and nephropathy was studied. In addition, examination was made of whether modifications of the treatment schedule could circumvent nephrotoxicity. Because preliminary studies had shown that female LOU/M/Wsl rats developed less doxorubicin-induced albuminuria, both male and female LOU/M/Wsl rats were treated with an iv dose of 1 mg doxorubicin/kg (body wt)/rat on five consecutive days and then weekly. Saline-treated animals served as controls. Albuminuria, serum albumin, and serum creatine levels were assessed weekly. For histologic examination, 5 male and 5 female rats were killed weekly. At day 14 and thereafter, doxorubicin-treated male rats showed albuminuria greater than or equal to 10 g/liter. Albuminuria of greater than or equal to 10 g/liter was not avoided by modifications of the treatment schedule. Female rats had on day 14 a urinary albumin level of 1.0-3.0 g/liter, yet reaching the level of greater than or equal to 10 g/liter at day 49. In male rats serum albumin levels decreased to levels below 10 g/liter (p less than .001 vs. finding for day 0); in contrast female rats maintained constant serum albumin levels till day 49. Serum creatine levels showed a tendency to rise, the values of male rats not being measured after day 28 due to hyperlipidemia; the levels of female rats increased from 37.8 +/- 3.0 mumol/liter to 53.7 +/- 2.5 mumol/liter on day 49 (P less than .001). At day 10 in male and female rats a grade 1-1.5 cardiomyopathy score, assessed according to the modified Billingham scoring system, was found, gradually increasing to grade 2.5-3 cardiomyopathy, both in males and females, on day 49. In male LOU/M rats the nephropathy developed steadily from day 14 and thereafter, whereas in females the rate of development of kidney damage was slower and at the end point of the study the severity of kidney lesions was less in comparison to that of the males. The onset of cardiomyopathy and nephropathy was simultaneous. It was concluded that cardiomyopathy observed in LOU/M rats is a phenomenon independent of nephropathy.

In a comparative study, the enzyme-linked immunosorbent assay, using peroxidase labeled anti-rat immunoglobulin M and immunoglobulin G, and the passive hemagglutination test were applied to determine the primary and secondary antibody response to lipopolysaccharide and tetanus toxoid in rats. In the enzyme-linked immunosorbent assay, the antigens were bound to the wells of polystyrene microplates, tetanus toxoid directly, and lipopolysaccharide after complexing it with methylated bovine serum albumin. After incubation with dilutions of the rat sera, the amount of antibody bound to the solid phase was quantified by means of peroxidase-labeled anti-immunoglobulin. The specificity of the enzyme immunoassay was tested by absorption of the sera with their respective antigens. The enzyme-linked immunosorbent assay proved to be more sensitive than the hemagglutination reaction, except when titers were determined during the secondary response to tetanus toxoid. Besides its specificity and sensitivity, the enzyme-linked immunosorbent assay is a convenient method for measuring both immunoglobulin M and immunoglobulin G antibodies. At low serum dilutions of lipopolysaccharide antisera, inhibition of the reaction in the enzyme-linked immunosorbent assay occurred. This phenomenon could be prevented by heating the sera at 56°C for 30 min. Lipopolysaccharide was immunogenic in rats over an extremely wide dose range (from 10 pg to 1 mg); the optimal immunogenic dose of lipopolysaccharide for young adult rats was 0.1 to 1,000 μg when administered intravenously, and that of tetanus toxoid was 5 to 10 lines of flocculation, as determined by the Ramon flocculation test.

To evaluate the functional significance of bis(tri-n-butyltin)oxide (TBTO)-induced thymus atrophy, lymphocyte depletion in spleen and lymph nodes, lymphopenia, and increased serum IgM and decreased IgG concentrations, in vivo and in vitro function studies were performed for specific and nonspecific resistance. Weaned male rats were fed diets containing 0, 20, or 80 mg TBTO/kg for at least 6 weeks. Regarding the thymus-dependent immunity, delayed-type hypersensitivity reactions to ovalbumin as well as tuberculin were significantly depressed at both dietary concentrations. Resistance to the nematode Trichinella spiralis was significantly suppressed as shown by a retarded expulsion of adult worms from the small intestine, increased counts of muscle larvae, reduced inflammatory reaction in parasitized musculature, and suppressed serum IgE titers. Also the secondary mercaptoethanol-resistant (presumably IgG) hemagglutinating antibody titer to sheep red blood cells was significantly reduced, while no significant alterations were found in IgM and IgG titers to T. spiralis, ovalbumin, and tetanus toxoid. TBTO exposure reduced the response of thymocytes in both treatment groups and of spleen cells in the 80-mg/kg group upon stimulation with T-cell mitogens and increased the response of spleen cells to B-cell mitogens. When calculated per whole spleen, the response to T-cell mitogens was strongly impaired but unaltered by B-cell mitogens. This difference can be explained by a relative increase of splenic B cells as a result of reduced numbers of T cells, as shown by cell surface marker analysis using monoclonal antibodies. Reduced splenic T-cell numbers appeared equally due to a decreased number of T helper and to T suppressor cells. From these data and from results of a time-sequence study in which effects of TBTO on cell count and cell viability of thymus, spleen, and bone marrow were investigated, it is concluded that TBTO-induced immunodeficiency was primarily due to its direct toxic action on thymocytes. When cultured in vitro in the presence of TBTO, viability of thymus and bone marrow cells was equally reduced, while after in vivo treatment viability of bone marrow cells was unaffected. Thus, the in vitro situation does not mimic the in vivo one. Concerning the nonspecific resistance, TBTO reduced macrophage function as shown by impaired splenic clearance of Listeria monocytogenes bacteria. From in vitro studies it is concluded that impaired in vivo splenic clearance was due to a reduction in both the number of adherent cells in the spleen and bacterial digestion on a cell for cell basis.(ABSTRACT TRUNCATED AT 400 WORDS)

After immunization of Wistar rats with different concentrations of ovalbumin (OA) emulsified in complete H37Ra adjuvant, the optimal antigen dose for specific IgM, IgG and IgE production and for delayed-type hypersensitivity appeared to be the same. It is therefore concluded that in the rat these function tests can be combined, which decreases the number of experimental animals needed. For the determination of IgM, IgG and IgE antibodies to OA, the enzyme-linked immunosorbent assay (ELISA) was used, which proved to be a specific method. In addition, using a multi-channel photometer, it is shown that specific antibody concentrations can be readily quantified by micro-ELISA instead of using end-point titrations.

We have investigated the effect of exposure to ozone on defense mechanisms to a respiratory infection with Listeria monocytogenes in the rat. For this purpose rats were continuously exposed to O3 concentrations ranging from 0.25 to 2.0 mg/m3 for a period of 1 week. In this model defense to a respiratory infection with Listeria depends on acquired specific cellular immune responses, as well as on natural nonspecific defense mechanisms. The results confirm earlier findings that show that ozone exposure can suppress the capacity of macrophages to ingest and kill Listeria. Moreover, the results show that ozone can also have a suppressive effect on the development of cellular immune responses to a respiratory Listeria infection, i.e., on T/B ratios in lung draining lymph nodes, delayed-type hypersensitivity responses to Listeria antigen, and lymphoproliferative responses in spleen and lung draining lymph nodes to Listeria antigen. The effects on the specific immune responses are especially overt if exposure to the oxidant gas occurs during an ongoing primary infection. The pathological lesions induced by a pulmonary Listeria monocytogenes infection were characterized by multifocal infiltrates of histiocytic and lymphoid cells. The foci sometimes had a granulomatous appearance. Moreover, the cellularity of the interstitial tissues was increased. In the lung many diffuse alveolar macrophages could be seen in the alveoli. Ozone exposure greatly increased the severity of the lung lesions and also of liver lesions resulting from the pulmonary infection. A prominent finding was the formation of granulomas in ozone-exposed and Listeria-infected rats. This increased severity of the lesions after ozone exposure and subsequent infection with Listeria was presumably not a result of additive ozone and Listeria-induced lesions, but rather an effect of ozone-induced impaired clearance of the bacteria, caused by depressed macrophage activity and cellular immunity. T-cell-dependent immune responses form an important component of defense to respiratory infections with bacteria and viruses, and possibly also to neoplasms. Since our study unequivocally shows an effect on T-cell-dependent immunity, ozone exposure has to be judged potentially hazardous with respect to such challenges of the lung.

Male and female Wistar rats were fed bis(tri-n-butyltin)oxide (TBTO) at 0, 5, 20, 80, or 320 mg/kg diet for 4 weeks. Clinical signs and decreases in feed and water consumption were observed in the 80 and 320 mg/kg groups. The serum transferase activities (alanine amino transferase and aspartate amino transferase were increased at 20 (males only), 80, and 320 mg/kg. The serum glucose and liver glycogen concentrations were lowered in the 320 mg/kg group. At 80 and 320 mg/kg the serum IgG level was reduced and IgM level was increased. Compared to controls the mean relative weight of the thymus was decreased at 20 (males), 80, and 320 mg/kg. In the groups receiving 80 or 320 mg/kg microcytic anemia was found. The white blood cell counts were decreased, due to the reduction in the number of lymphocytes in the 80 (males) and 320 mg/kg groups. The concentration of neutrophilic granulocytes was increased in the highest dose group. Histopathologic effects included a dose-related lymphocyte depletion of thymic cortex and of T lymphocytes in spleen and mesenteric lymph nodes. In the spleen also depletion of iron stores was found, and in the medullary sinuses of mesenteric lymph nodes, rosettes of erythrocytes were found around mononuclear cells; the occurrence of rosettes increased with dose from 5 to 80 mg/kg, and appeared to be the most sensitive parameter. A low incidence of areas of liver necrosis with inflammatory reaction and bile duct hyperplasia was found in the 320 mg/kg group. A viral or bacterial etiology could be demonstrated for these liver lesions, but they appeared associated with TBTO-induced ulcerative inflammation of the common bile duct as shown in an additional study. In 6-week studies exposure of male weanlings to the 0, 20, and 80 mg/kg diets, the serum insulin concentration in the treated groups was decreased, although the response to glucose challenge was unaffected. The serum thyroxin and thyrotropin (TSH) concentrations were reduced, whereas the luteinizing hormone (LH) concentration was increased in the 80 mg/kg group. The concentrations of follicle-stimulating hormone (FSH) and corticosterone were not changed. The release of LH and FSH was enhanced in the 80 mg/kg group and a tendency toward reduced release was found for TSH. Using immunocytochemistry a dose-related reduction was found in the number and staining intensity of TSH-producing cells in the pituitary, correlating with histopathologically decreased activity of the thyroid.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies in various animal models indicate that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and approximate isostereomers produce a similar pattern of toxic responses, including thymic atrophy. Effects on the immune system include suppression of cell-mediated immunity. Also in man, exposure to TCDD-related halo-genated aromatic hydrocarbons has been shown to impair thymus-dependent immunity. Several investigations have been performed to clarify the thymus atrophy. TCDD is not directly toxic for thymocytes, nor does it act through an increase of serum corticosteroid levels or a decrease of growth hormone levels. In studies with murine inbred strains differing in sensitivity to TCDD it has been shown that thymic atrophy segregates with the Ah locus and thus appears to be mediated by the Ah receptor. Recent studies indicate that TCDD acts through a receptor in thymic epithelium. Treatment of monolayers of thymic epithelial cells with TCDD resulted in the suppression of epithelium-dependent responsiveness of co-cultured thymocytes to mitogens. These results provide direct evidence that thymic epithelium is a target for TCDD. These findings are consistent with the hypothesis that TCDD stimulates terminal differentiation of reticular thymic epithelial cells to a stage at which they have lost their ability to support thymocyte maturation, thus leading to depletion of cortical thymocytes.

Summary A total of 45 normal or enlarged thyroid glands of adult slaughter cattle were weighed and analyzed by thin‐layer chromatography for the presence of residues of the following thyreostatic compounds: thiouracil, methylthiouracil, propylthiouracil, phenylthiouracil, and methimazole. In 21 glands, mostly from imported animals, residues of methylthiouracil or methimazole were detected. These thyroids ranged in weight from 68–245 g (mean value 118 g). Glands in which no residues were found, varied in weight between 20 and 124 g (mean value 42 g). Taking an upper limit of 60 g as normal, 4 out of 22 were ‘false‐positive’ with respect to the 5 thyreostatic compounds screened for, while no false‐negative cases were scored. On the basis of these results the weight increase of the thyroid gland is proposed as a simple indirect parameter in the screening for the illegal use of thyreostatic compounds in slaughter cattle.