Acute testicular toxicity of 1,3-dinitrobenzene and ethylene glycol monomethyl ether in the rat: evaluation of biochemical effect markers and hormonal responses.

The studies described in this paper were undertaken to evaluate the use of plasma enzymes of testicular origin and plasma hormones as markers of acute testicular toxicity. Rats were dosed by gavage with a single dose of either 1,3-dinitrobenzene (1,3-DNB) or ethylene glycol monomethyl ether (EGME). Two experimental designs were used: a dose response and a time-dose response course. Lactate dehydrogenase isozyme C4 (LDH-C4) and sorbitol dehydrogenase (SDH) were used as germ cell markers and leucine aminotransferase (LAT) and androgen binding protein (ABP) were used as Sertoli cell markers. Luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone were also monitored. Histopathology confirmed the known testicular toxicity of 1,3-DNB and EGME. 1,3-DNB induced Sertoli cell damage with associated degenerative changes in late pachytene spermatocytes. The effects of EGME were mainly on early and late pachytene and dividing spermatocytes. No changes in either testicular or plasma SDH or LAT were found. Similarly no effects were observed for plasma LH or testosterone. However testicular LDH-C4 and testosterone, plasma LDH-C4, ABP, and FSH did show compound related effects. LDH-C4 was reduced in testis and increased in plasma with both compounds and plasma LDH-C4 remained elevated up to 14 days after dosing. ABP levels in plasma were increased with 1,3-DNB and EGME. A reduction in testicular testosterone levels was recorded and plasma FSH concentrations were elevated after EGME treatment. It is concluded that plasma LDH-C4 activity and ABP may be of diagnostic value in acute testicular toxicity. Increases in plasma LDH-C4 precede noticeable histological findings.

The in vitro effects of four isomers of dinitrotoluene on rat Sertoli and Sertoli-germ cell cocultures: germ cell detachment and lactate and pyruvate production.

The present study was undertaken to evaluate the in vitro effects of four isomers of a known testicular toxicant, dinitroluene (DNT). Rat Sertoli or Sertoli-germ cell cocultures were treated, after 3 days in culture, with DNT isomers (0.01 to 100 microM) or 1,3-dinitrobenzene (1,3-DNB) for 24 hr. Cellular morphology, germ cell detachment (GCD) and lactate pyruvate production were used as sensitive effect markers of in vitro toxicity. Morphologically the Sertoli cell monolayer remained intact 24 hr after exposure to DMSO, 1,3-DNB, or DNT isomers. Some apparent cytotoxicity was observed at 100 microM 3,4-DNT: the monolayer was disrupted with extensive vacuolation of the Sertoli cells. Cocultures treated with concentrations of 50 microM DNT isomers closely resembled cells treated with 100 microM 1,3-DNB. GCD increased in a dose-dependent manner (0.01 and 10 microM DNT isomers) increasing between 2- and 10-fold over control. Both lactate and pyruvate production increased with rising concentrations of DNT isomers. The most sensitive effect was seen with 3,4-DNT (10 to 25 microM). In the case of 2,6-DNT, despite increases in GCD and lactate production, only a minimal increase in pyruvate was demonstrated. Overall, the ratio of lactate to pyruvate production declined with increasing doses of DNT. These results indicate that the four isomers of DNT directly affected Sertoli cell morphology and function, effects comparable to those seen with the Sertoli cell toxicant 1,3-DNB. Further, the data support the hypothesis that DNT may be a Sertoli cell toxicant.

Acute in vitro thyrotropin regulation of lysosomal enzyme activity in the thyroid follicular cell.

The acute effect of a physiological concentration (1 mU/l) of thyrotropin (TSH) on the activity of four lysosomal enzymes in the thyroid follicular lining cell has been studied by quantitative cytochemical techniques. N-acetyl-beta-glucosaminidase (NAG) activity was increased by 14% after 10 min TSH stimulation and NAG and beta-galactosidase activities were increased by 24% and 25% respectively (P less than 0.05) after 20 min stimulation and by 40% and 45% (P less than 0.05) respectively after 30 min stimulation with TSH, indicating an early processing of these carbohydrate residues in thyroglobulin. Acid phosphatase activity, an acid hydrolase unrelated to the hydrolysis of thyroglobulin, was unchanged 30 min after TSH stimulation. Leucyl-beta-naphthylaminidase (LNA) activity changed biphasically with peak activities of 7 and 25 min possibly representing an early fusion of endocytotic vesicles and lysosomes and later the release process of the thyroid hormones. The changes in LNA activity and thus membrane permeability were not reflected in the other enzyme activities studied. This may indicate that the TSH regulation of lysosomal enzyme activities could be independent to the endocytotic process, which is known to involve fusion of lysosomes and endocytotic vesicles. In conclusion we have demonstrated for the first time with physiological concentrations of TSH a specific acute regulation of some lysosomal enzyme activities which may be involved in thyroglobulin processing. Further, these effects may be independent of the changes in lysosomal membrane permeability due to formation of secondary lysosomes.

Assessment of the bioactivity of human and bovine thyrotrophin preparations using a porcine thyroid cell bioassay.

The biopotency of six preparations of thyrotrophin (TSH) has been compared in a highly sensitive in vitro porcine thyroid cell bioassay using iodide uptake as an endpoint. Three of these preparations were of human origin and three derived from bovine pituitary tissue. One human TSH preparation, the 2nd International Reference Preparation, 80/558, was used to calibrate the other five. The log dose-log response curves for all preparations were sigmoidal in shape. For the purpose of evaluation the central linear portions of the curves were compared. With all preparations the slopes in this region were very similar. The relative biopotencies of the bovine preparations (unit:unit) were at least five times those of the human standards when measured using the porcine thyroid cell bioassay. These findings emphasise the need to control the TSH standards employed in a variety of bioassays, particularly those used for between-laboratory comparison.

Acute stimulation of thyroidal NAD+ kinase, NADPH reoxidation, and peroxidase activities by physiological concentrations of thyroid stimulating hormone acting in vitro: a quantitative cytochemical study.

Quantitative cytochemical techniques have been employed in a study of some of the acute effects of low doses (0.01----1 mU/liter) of TSH on the metabolism of guinea pig thyroid segments maintained in nonproliferative organ culture. The enzymes involved in the synthesis of NADP+ (NAD+ kinase), its reduction by the pentose-shunt (glucose 6-phosphate dehydrogenase), and its reoxidation both by the microsomal electron chain (diaphorase activity) and by participation in other cellular processes, have been examined. The effect of TSH on peroxidase activity has also been studied. After 10 min stimulation with TSH (1 mU/liter) there was a 60% increase in NAD+ kinase activity which preceded changes in the microsomal reoxidation of NADPH (up 33% by 30 min). There were no changes in the activity of glucose 6-phosphate dehydrogenase. There was a sustained rise in peroxidase activity which reached 129% over control after 30 min. This is the first in vitro demonstration of an acute stimulation of peroxidase and kinase activities by physiological concentrations of TSH. NADPH reoxidation after stimulation with TSH was such that the ratio of NADPH reoxidized via the microsomal respiratory pathway (diaphorase, hydrogen pathway 1) relative to that available for cytosolic utilization (hydrogen pathway 2) increased compared to the unstimulated controls. We suggest that increased NADP+ production (via NAD+ kinase activity) and the preferential shuttling of the NADPH for reoxidation via the microsomal respiratory pathway, coupled with greatly stimulated peroxidase activity, may be important regulators of the control of thyroglobulin iodination and hence thyroid hormone production.

Assessment of the biopotency of anti-thyroid drugs using porcine thyroid cells.

The effect of six drugs on the uptake and organification of iodide by porcine thyroid cells stimulated with bovine TSH (10 miU/L) has been investigated. The drugs fall into two categories: the peroxidase inhibitors, methimazole (MMI), 2-thiouracil (2-TU) and 3-amino 1,2,4 triazole (3-ATA) and the ionic inhibitors, lithium chloride (LiCl), potassium perchlorate (KC10(4], and sodium iodide (NaI). All the drugs led to a dose-related inhibition of iodide metabolism. The most potent effect on iodide uptake was seen with NaI which inhibited this function by 20% even at 10(-8) mol/l. In contrast, the most potent effect on iodide organification was observed with methimazole which led to a 25% inhibition at 10(-8) mol/l. The concentrations of drug which gave rise to a 50% inhibition of iodide uptake were (mumol/l) 0.26 (NaI), 3.5 (KClO4), 9.7 (2-TU), 15 (MMI), 26 (3-ATA), and 1500 (LiCl). The comparable figures for organification were 0.13 (MMI), 0.18 (2-TU), 0.23 (NaI), 1.2 (3-ATA), 15 (KClO4) and 1300 (LiCl). We conclude that this in vitro system has considerable potential for the assessment of potency and possible bioassay of anti-thyroid drugs of varying structures and sites of action.

Protein-A purified human immunoglobulins: a comparison of thyroid stimulating and thyrotrophin receptor binding activities in thyrotoxicosis.

Protein-A purified human thyroid stimulating immunoglobulins (TSIg) and thyrotrophin binding inhibiting immunoglobulins (TBIIg) were measured in euthyroid subjects and thyrotoxic patients by bioassay and TSH radioligand receptor assay respectively. Unextracted sera from euthyroid and thyrotoxic subjects inhibited both basal and TSH stimulated iodide uptake in the bioassay, which was based on iodide uptake in porcine thyrocytes. Similar effects were seen with Ig and TSIg extracted from sera using either polyethylene glycol or ammonium sulphate. However IgG and TSIg prepared using Protein-A Sepharose CL-4B from sera of euthyroid subjects had little effect in this system. The majority of Protein-A purified TSIg preparations from sera of thyrotoxic patients stimulated iodide uptake in procine thyrocytes in a dose-dependent manner and most (85%) diluted parallel to both bovine and human TSH. TSIg and TBIIg from 73 patients with thyrotoxicosis were assessed using the bioassay and receptor assay and compared to a control group of 35 euthyroid subjects. The median (and range) values for TSIg and TBIIg in the euthyroid group were 4.35 (0.8 to 7.5, % stimulation over control) and 2.7 (-9.3 to 8.6, TBII index) for the bioassay and radioreceptor assay respectively. A value of greater than 10.0 in both assays was taken as a positive result. Of the thyrotoxic patients 61 out of 73 were positive in the bioassay (83.6%) compared to 60 in the radioreceptor assay (82.2%). There was a positive correlation between the two assays (r = 0.821, P less than 0.001). Of the 73 thyrotoxic patients 40 were untreated, 18 had received carbimazole and 15 had been previously treated with iodine-131. TSIg levels in the untreated thyrotoxics were similar to those in either group of treated patients. However they were higher (P less than 0.05) in the iodine-131 group than in the patients treated with carbimazole. Similar results were obtained for TBIIg. The coupling of a specific extraction method for human serum IgG with a bioassay for TSIg has demonstrated a high prevalence of these immunoglobulins in patients with thyrotoxicosis. The agreement between this assay and a radioreceptor assay was good, indicating that TSH displacing and thyroid stimulating activities of these immunoglobulins are closely related.

Measurement of low concentrations of bovine thyrotrophin by iodide uptake and organification in porcine thyrocytes.

Thyrocytes isolated from porcine thyroids by mechanical and enzymatic dispersion and cultured in Eagle's minimal essential medium, supplemented with 5% (v/v) fetal calf serum, glutamine and cortisol, formed a continuous monolayer within 48 h. This monolayer was without cytochemical peroxidase and diaphorase (NADPH reoxidation) activity. In the presence of bovine thyrotrophin (bTSH; 50 mu./l) the cells developed a follicular-like architecture which was maximal at 4 days before reverting back to a uniform monolayer at 6 days. There were no detectable changes in the total DNA content over this period. The follicular structures had marked diaphorase and peroxidase activity, the latter being apically distributed. Concomitant with follicle formation bTSH induced uptake and organification of iodide presented to the cells during the last 6 h of culture. The extent of this process depended on the dose of bTSH and the duration of stimulation. The most sensitive effects for both iodide uptake and organification occurred with 1 mu. bTSH/l and were maximal with 100 mu./l. Uptake and organification were increased 20 +/- 8-fold and 9.6 +/- 2-fold (n = 10) respectively over the control with 100 mu./l and the doses of bTSH at which a half maximal response was seen (ED50) were 15 +/- 2 and 7 +/- 1 (S.D) mu./l (n = 10) respectively. On changing the culture medium to a serum-free system using HB101 culture medium the stimulation time for the most sensitive bTSH effect was reduced to 2.5 days.(ABSTRACT TRUNCATED AT 250 WORDS)

On the biopotency and site of action of drugs affecting endocrine tissues with special reference to the anti-steroidogenic effect of anaesthetic agents.

Dispersed guinea-pig adrenal cells or mouse Leydig cells were stimulated with a saturating dose of adrenocorticotrophin (ACTH, 50 ng/1) or luteinizing hormone (LH, 5IU/1), respectively. The incubations were performed in the presence of increasing concentrations (10(-9) - 5 X 10(-4)mol/l) of the anaesthetic agents propofol, thiopentone and etomidate. At the end of this stimulation period, cortisol (from the adrenal preparation) or testosterone (from the Leydig cell culture) were assayed by radioimmunoassay. Propofol, thiopentone and etomidate all inhibited ACTH-stimulated cortisol secretion in a dose-related fashion. Similar inhibition of LH-stimulated testosterone output was found with propofol and thiopentone whereas etomidate was without effect at any concentration employed, an observation in accordance with its known site of action, 11 beta-hydroxylase, an enzyme which is not involved in the biosynthesis of testosterone. The concentration (mumol/l) of anaesthetics which gave 50% inhibition (ED50) of ACTH-stimulated cortisol secretion was 0.1 +/- 0.002 (n = 7), 160 +/- 18 (n = 3) and 170 +/- 18 (n = 3) (mean +/- s.e.m.) for etomidate, thiopentone and propofol, respectively. The corresponding values for the LH stimulated testosterone output from the Leydig cell preparations were 186 (thiopentone) and 180 (propofol) mumol/l. In a separate series of experiments adrenal cells were stimulated with (a) the cortisol precursor steroids (all at 10(-5)mol/l) pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone and 11-deoxycortisol, (b) dibutyryl cAMP (10(-3)mol/l) or (c) ACTH (100 ng/l) in the presence and absence of either etomidate (5 X 10(-5)mol/l), propofol (2.5 X 10(-4)mol/l) or thiopentone (5 X 10(-4)mol/l). All the stimulators increased cortisol production by > 7-fold over that seen in their absence. Propofol depressed ACTH and dibutyryl cAMP induced cortisol output by > 60% (P < 0.05) but was without effect when the steroid precursors were used, suggestive of an inhibition between the sequence involving ACTH binding -> pregnenolone production. In contrast, etomidate and thiopentone reduced cortisol secretion by > 40% (P < 0.05) regardless of the stimulator used, indicating that at least one site of action was at the level of the final enzymic step of cortisol synthesis, i.e. 11beta-hydroxylase.

Microheterogeneity of luteinizing hormone in pituitary glands from women of pre- and postmenopausal age.

Luteinizing hormone from the pituitaries of women of premenopausal (Pre-M) and postmenopausal (Post-M) age has been studied to further understand the variations in the quality of plasma LH in women of different ages. The median and range for bioactive LH was 101: 85-155 IU/mg protein and 83: 52-201 IU/mg protein for the Pre- and Post-M groups respectively. The ratios of LH bioactivity/immunoreactivity (B:I) were 1.04 +/- 0.13 (Pre-M) and 0.97 +/- 0.02 (Post-M). Broad range isoelectrophoretic profiles of the pituitary extracts revealed a heterogeneous population of coincident bioactive and immunoreactive LH peaks. Recoveries from the columns were similar in both groups and B:I values of the LH material throughout the columns were between 0.5 and 2.0 in over 90% of the fractions. Between 31.8 and 36.6% of the LH from both groups appeared between pH 7 and pH 8. The most striking difference between the two groups was that greater than 34% of the bioactive LH from the Post-M pituitaries, but less than 6% of the LH from the other group had migrated as a major discrete peak to an acidic region of pH less than 6. We conclude that although there is no difference in total LH activity in the pituitaries from the two groups, substantially more 'acidic' LH material is stored in the postmenopausal pituitaries. These observations are consistent with the recent finding of greater circulating levels of acidic LH in plasma from post-menopausal women.

Negative feedback effects on ACTH secretion by cortisol in Cushing's disease.

Bioassayable plasma ACTH and corticosteroid levels were measured during constant infusions of low doses of cortisol (3-15 mg/h) into normal subjects and into two patients with Cushing's disease. Plasma ACTH levels decreased significantly in all subjects within 60 min from the start of the infusion. The rate of rise of plasma corticosteroid levels concomitant with this feedback suppression were considerably higher in the patients with Cushing's disease (greater than 13 nmol/l/min) than in normal subjects (3-6 nmol/l/min). In one of the Cushing's patients, ACTH secretion resumed in spite of the corticosteroid levels rising at a rate of greater than 20 nmol/l/min. It is concluded that cortisol-induced suppression of ACTH secretion may be operational in Cushing's disease but that the mechanism is less sensitive than in normal subjects.

Negative rate-sensitive feedback effects on adrenocorticotrophin secretion by cortisol in normal subjects.

Plasma ACTH and corticosteroid levels were measured in normal subjects during constant infusion of either 0.9% (W/V) NaCl solution or cortisol, and during insulin-induced hypoglycaemia. During infusions of 0.9% NACl solution the secretion of ACTH and corticosteroids was episodic. Fast, rate-sensitive, negative feedback inhibition of ACTH secretin was observed during cortisol infusions, when the corticosteroid levels were within the physiological range (200-750 nmol/l) and were rising at a rate of between 5 and 10 nmol/l per min for 30 min or longer. When plasma corticosteroid levels were in a steady state, the initial fast feedback effects were abolished and ACTH secretion resumed. However, this recovery of ACTH secretion was not seen when the corticosteroid levels were persistently above 800 nmol/l. It appears that corticosteroid-induced negative feedback in man may be both rate- and level-sensitive. During insulin stress test ACTH secretion fell at time when the plasma corticosteroid level was rising rapidly (greater than 5 nmol/1 per min) despite persistent hypoglycaemia.

Effect of human plasma on the bioactivity of adrenocorticotrophin.

The effect of plasma on the bioactivity of ACTH was investigated with the cytochemical section bioassay. Levels of ACTH in normal human plasma were underestimated if the plasma concentration in the incubation medium exceeded 2% and if the plasma ACTH level was less than 2 ng/l. A retrospective analysis of 250 plasma ACTH assays revealed that the ACTH concentration was consistently underestimated in the 1:100 dilution compared with the 1:1000 dilution, although not to the extent that any assay would be rejected for non-parallelism. The extent of the underestimation increased as the ACTH concentration increased. Guinea-pig adrenal sections responded faster to 1:10 or 1:50 dilutions of human plasma (containing 2 ng ACTH/l) than to either 1:100 or 1:1000 dilutions of that same plasma, or to ACTH (over the range 0.005-5 ng/l)in the absence of plasma. This faster response could also be seen in the absence of plasma by increasing the ACTH concentration above 5 ng/l. It seems that plasma contains factors which potentiate the biological effect of ACTH in the cytochemical assay system.U

The use of an inexpensive, general purpose microcomputer in quantitative cytochemistry.

A 32K Commodore Pet microcomputer has been interfaced with two Vickers microdensitometers. This system allows for the simultaneous logging of data from two densitometers being operated independently of each other. Software for the statistical analysis of data generated by the densitometers, and for use with the cytochemical bioassay of hormones, is described. The densitometer/Pet system is relatively cheap, reliable, and readily adaptable to other applications.