The mutagenic potential of acetonitrile in the bone marrow and peripheral blood of the mouse.

Acetonitrile was tested for its ability to induce clastogenic or aneugenic effects through the induction of micronucleated polychromatic erythrocytes (MNPCE) in mouse bone marrow and peripheral blood. Groups of NMRI mice, five males and five females, were administered a single i.p. dose of acetonitrile, corresponding to the maximum tolerated dose (MTD), 100 or 125 mg/kg body wt for males and females, respectively. Bone marrow was sampled at 18, 24 or 36 h after treatment, while peripheral blood was sampled before and 24, 48, 72 and 96 h after treatment. Positive controls were administered cyclophosphamide (65 mg/kg i.p.). Acetonitrile did not increase the incidence of MNPCE in either bone marrow or peripheral blood in male mice or in peripheral blood in females. A small, but statistically significant (P: < 0.05), increase was observed in female bone marrow 36 h after administration, but since this was within the range of the control data it is not considered to be of biological significance. Cyclophosphamide increased the incidence of MNPCE in bone marrow and peripheral blood of both sexes. It is concluded that acetonitrile is neither clastogenic nor aneugenic in the bone marrow of the mouse at the MTD.

Developmental toxicity of di-(C(7)-C(9) alkyl) phthalate and di-(C(9)-C(11) alkyl) phthalate in the rat.

Two phthalate esters, di-(C(7)-C(9) alkyl) phthalate (D79P) and di-(C(9)-C(11) alkyl) phthalate (D911P), have been assessed for their potential to cause developmental toxicity in the rat. Groups of 22 timed-mated Sprague-Dawley rats were administered 250, 500, or 1000 mg/kg D79P or D911P daily by oral gavage (5 ml/kg) between gestation days (GD) 1 and 19. Control animals received the vehicle (olive oil) alone. On GD20, the animals were sacrificed and the fetuses examined. Treatment resulted in no signs of maternal toxicity, as assessed by adjusted maternal bodyweight gain throughout gestation and clinical examinations, and no effects upon litter size, fetal survival or bodyweight. Pups of the high dose D79P and intermediate and high dose D911P groups showed increased incidences of supernumerary lumbar ribs. There was a significant increase in dilated renal pelves in pups of the low dose D79P and high dose D911P groups, but only for D911P was there a significant trend. Consequently, the no observed adverse effect level (NOAEL) for maternal toxicity for both D79P and D911P is 1000 mg/kg/day. The NOAEL values for developmental toxicity are 500 mg/kg/day D79P and 250 mg/kg/day D911P.

Two-generation reproduction toxicity studies of di-(C(7)-C(9) alkyl) phthalate and di-(C(9)-C(11) alkyl) phthalate in the rat.

Di-(C(7)-C(9) alkyl) phthalate (D79P) and di-(C(9)-C(11) alkyl) phthalate (D911P), based on high-normality linear oxo-alcohols, have been assessed for their impact upon reproductive performance in Sprague-Dawley rats. Rats were continuously exposed to either D79P or D911P at dietary levels of 0%, 0.1%, 0.5%, or 1.0% over two generations. Selected F(0) offspring (F(1) generation) were exposed to the same dietary concentration of D79P or D911P as the respective F(0) animals, and were mated to produce F(1) offspring. Both D79P and D911P markedly reduced body weight gain in F(0) and F(1) adult males at the highest dose, but females were affected to a lesser extent. There was no impairment of fertility, fecundity, or development in either generation, but body weights of offspring in the 1.0% D79P and 1.0% D911P groups were slightly and transiently reduced over the weaning period. Although decreases in the weight of several organs were accounted for by depressed body weight, ovary weights were reduced in both generations exposed to 1.0% D79P, and epididymidal weights were slightly reduced in adults of both generations exposed to 1.0% D911P. However, ovarian function-assessed by the oestrus cycle and mating behaviour-and epididymidal sperm concentration, motility, and morphology were unaffected by either substance. Treatment resulted in liver changes, particularly in males, characterised by increased liver weight in young animals, histopathologic changes and reduced organ weight in mature animals, and an increase in palmitoyl CoA oxidase activity. In conclusion, neither D79P nor D911P impaired reproductive function in rats when administered in the diet at levels that induce systemic toxicity, and the NOAEL for effects on reproduction in the rat is 0.5% for both D79P and D911P.

The oestrogenic potential of the phthalate esters.

The phthalate esters represent a class of chemicals used widely and diversely in industry. Concern that phthalates might be oestrogenic arose from observations that the diesters inhibited the binding of 17beta-estradiol to isolated oestrogen receptors and stimulated the expression of cellular oestrogen-sensitive endpoints (gene expression, mitosis) in vitro. However, conflicting results have been found in comparable studies, and those studies that have demonstrated oestrogen mimicry have generally done so at concentrations approaching, or above, the limit of water solubility for the phthalates. The monoesters (the primary metabolites of the diesters in vivo) are inactive in similar in vitro tests. Furthermore, the diesters have not shown any oestrogenic activity in numerous and diverse studies in vivo at doses eliciting systemic toxicity. Consequently, the oestrogenic activity of phthalates identified in in vitro studies is not relevant to humans or the environment.

The distribution, metabolism and function of creatine in the male mammalian reproductive tract: a review.

Creatine is widely distributed throughout the male reproductive system in several mammalian species, and proteins involved in its metabolism and transport have been reported in a number of cell and tissue types. Creatine is synthesized within some organs, incorporating nitrogen from amino acid metabolism. Although creatine metabolism is obligatory for the motility of sea urchin spermatozoa, this does not appear to be the case for mammals. The possible functions of creatine within the reproductive tract are discussed.

Creatine metabolism in the seminiferous epithelium of rats. I. Creatine synthesis by isolated and cultured cells.

The testis synthesizes creatine from both arginine and glycine precursors, but when rat testicular tissue is separated into seminiferous tubules and interstitial cells, creatine synthesis occurs only in the tubular fraction. The purpose of the work presented here was to define the locus of creatine synthesis within the seminiferous tubules, by using cell separation and culture techniques to examine synthesis in the Sertoli cells and germ cells. The total creatine content, in the cellular compartment and incubation medium, of Sertoli-germ cell co-cultures and of Sertoli cell-enriched cultures, largely free of germ cells, increased by similar amounts over a 24 h incubation period. Sertoli cell-enriched cultures incorporated radioactivity from L-[guanidino-14C]arginine and [1-14C]glycine into both creatine and its biosynthetic precursor, guanidinoacetic acid. Isolated germ cells did not incorporate radioactivity from L-[guanidino-14C]arginine into either creatine or guanidinoacetic acid when incubated at a similar density and protein concentration under similar conditions. It is concluded that the synthesis of creatine observed in isolated rat seminiferous tubules occurs within the Sertoli cells and not the germ cells.

Creatine metabolism in the seminiferous epithelium of rats. II. Effect of modulators of cellular biochemical function on creatine secretion by cultured Sertoli cells.

The Sertoli cells have been identified as the primary locus for creatine synthesis within the seminiferous epithelium. The purpose of the studies reported here was to examine the effect of modulators of Sertoli cell function on creatine secretion by primary cultures of these cells. Sertoli cell-enriched cultures, maintained in a defined medium, secreted creatine into the incubation medium in a manner that was linear with time over at least 6 h, but which had reached a plateau within 24 h. Secretion was stimulated by physiological and toxicological modulators of Sertoli cell function. Incubation of Sertoli cell-enriched cultures in the presence of FSH (> or = 40 mU ml-1), dibutyryl cyclic AMP (> or = 0.1 mmol l-1), mono-(2-ethylhexyl) phthalate (> or = 1 mumol l-1) or cadmium (> or = 3 mumol l-1) increased the secretion of creatine into the incubation medium by at least 85% over 24 h. Creatine secretion by Sertoli cell-enriched cultures, incubated over 4 h in a balanced salt solution, was independent of exogenous L-glutamine. However, the stimulation of secretion induced by 1 mmol dibutyryl cyclic AMP l-1 was dependent on the presence of 4 mmol L-glutamine l-1 in the incubation medium, which suggests that an increase in creatine secretion occurs as a consequence of stimulated glutamine oxidation.

Effect of triglycyl-lysine-vasopressin on skin blood flow and blood loss during wound excision in patients with burns.

Excisional therapy often results in large-volume blood loss. Triglycyl-lysine-vasopressin selectively decreases dermal blood flow and therefore was tested for efficacy in limiting intraoperative blood loss in a series of patients undergoing excisional therapy. Ten patients with symmetric injuries were treated with intravenous triglycyl-lysine-vasopressin after excision of half of their burn wound. Blood loss, which was quantified by weighing sponges used to absorb shed blood, was significantly decreased after treatment. Triglycyl-lysine-vasopressin treatment was safe and effective and should be considered in cases when large-volume blood loss is expected.

Correlation of the local and systemic cytokine response with clinical outcome following thermal injury.

Eighty-eight patients with acute thermal injury were evaluated. Forty-eight hours after injury, TNF, IL-6, and IL-8 were significantly present in the systemic circulation, lung, normal skin, and thermally injured skin. The presence of TNF, IL-6, and IL-8 proteins in the lung, normal skin, and thermally injured skin were associated with TNF, IL-6, and IL-8 mRNA upregulation. Logistic regression analysis controlling for the Abbreviated Burn Severity Index demonstrated that the presence of IL-8 in the lung was associated with early pulmonary physiologic dysfunction (p = 0.006) and nosocomial pulmonary infection (p = 0.040). We conclude that acute thermal injury initiates an early systemic, lung, and skin response involving TNF, IL-6, and IL-8. The TNF, IL-6, and IL-8 protein present in the lung and skin in response to acute thermal injury are generated locally and do not originate from the systemic cytokine pool. The lung cytokine response to acute thermal injury may initiate local organ failure.

Urinary creatine profiles after administration of cell-specific testicular toxicants to the rat.

Cell-specific testicular toxicants have been used to examine the distribution of creatine within the rat testis, and to examine the potential use of creatinuria as a non-invasive indicator of testicular damage. Groups of rats were administered single doses of various toxicants: a germ cell toxicant (methoxyacetic acid, MAA), one of two Sertoli cell toxicants (di-n-pentyl phthalate, DPP or 1,3-dinitrobenzene, 1,3-DNB), or a Leydig cell toxicant (ethane-1,2-dimethane sulphonate, EDS). Urinary creatine and creatinine levels were monitored in 24 h periods over the following 48 h, after which time the testes were removed, weighed and, after processing, sections were examined by light microscopy. All four treatments resulted in reductions in relative testis weight (RTW) and produced morphological changes similar to those which have been previously reported. In addition, MAA, DPP and 1,3-DNB all caused significant elevations in urinary creatine excretion and the urinary creatine:creatinine ratio (Cr/Crn) within 24 h. EDS had no such effect. We conclude that creatine is associated with the cells of the seminiferous epithelium, and that elevated urinary excretion of creatine may serve as a non-invasive marker for damage to these cells in vivo.

Secretion of inhibin beta A by endoderm cultured from early embryonic chicken.

Although several reports have indicated a role for endoderm in the regulation of heart development, the mechanism remains unknown. To begin characterization of endoderm-secreted proteins, explants from postgastrulation (Hamburger-Hamilton stage 5/6-8) chicken embryos were cultured in defined medium. Fluorography of SDS-PAGE gels revealed a pattern of synthesized, secreted proteins that was independent of time in culture or embryonic stage when explants were removed. Approximately 10 labeled bands were detected, the most prominent of which migrated at 17, 25, and 200 kDa. ELISA analysis revealed that while acidic and basic fibroblast growth factor-like antigens were barely detectable, fibronectin and inhibin beta A were very reactive. Western blot analysis verified the presence of fibronectin and, most remarkably, inhibin beta A, activin dimers of which have recently been implicated in Xenopus mesoderm induction (Smith, Price, Van Nimmen, and Huylebrock (1990). Nature 345, 729.)

Localization of bFGF-like proteins as punctate inclusions in the preseptation myocardium of the chicken embryo.

Immunocytochemistry has been employed to map the appearance of bFGF-like proteins in precardiac and preseptation myocardial cells between stages 6 and 15 of chicken embryogenesis. Stage 6 embryos exhibited no staining, with the exception of a subtle signal in endoderm cells. At subsequent stages, staining was observed only in cells of the developing myocardium, first appearing at the time of heart tube fusion (stage 9+) as punctate cytoplasmic aggregates. While the expression of bFGF-like antigen was temporally similar to that of myosin heavy chain, their staining patterns differed in that bFGF-like proteins were nonsarcomeric and did not extend into the inflow or outflow tracts. Western blotting of heparin agarose affinity-isolated proteins from stage 15 hearts revealed an antigen migrating at approximately 19 kDa. In contrast with the unique localization of bFGF-like proteins in myocardial cells, FGF receptor (FGFR) staining was widely distributed in the embryo; however, concentrated deposits of FGFR were detected in endothelial and myocardial cells, which diminished in the myocardium but not in the endothelium by stage 15. These results suggest that FGF-like proteins may have autocrine and/or paracrine functions during early cardiac morphogenesis.

Leukopenia in acute thermal injury: evidence against topical silver sulfadiazine as the causative agent.

White blood cell data from time of admission to 4 days after burn injury was retrospectively reviewed to determine differences in the incidence of leukopenia in patients with burn injuries treated topically with either silver sulfadiazine or silver nitrate. WBC counts decreased in both groups of patients during the first 3 days after burn injury. An incidence of leukopenia (WBC count less than or equal to 5000/mm3) was observed in of 40 (47.5%) patients treated with silver sulfadiazine and in 13 of 30 (43.3%) patients treated with silver nitrate. There was no statistical difference in the incidence of leukopenia between the two treatment groups. These data suggest that silver sulfadiazine may not be the cause of the leukopenia observed early after burn injury.

Effect of chemical modification of histidine and tyrosine residues in toxic shock syndrome toxin 1 on the serologic and mitogenic activities of the toxin.

Modification of three or four of the five histidine residues in the toxic shock syndrome toxin 1 (TSST-1) with diethylpyrocarbonate did not inhibit the precipitin reaction of the modified TSST-1 with polyvalent antisera to the toxin. Monoclonal antibody 7T did not react with the modified TSST-1, but monoclonal antibody 8T did react with the toxin. Up to 50% of the mitogenic reaction of TSST-1 was inhibited by the histidine modification. Modification of one or two of the nine tyrosine residues in TSST-1 did not inhibit the precipitin reaction with polyclonal antisera to the toxin but did inhibit 85% of the mitogenic reaction.

Determination of biologically active region in toxic shock syndrome toxin 1.

The biologically active areas of toxic shock syndrome toxin 1 (TSST-1) were investigated using chemical fragmentation of the toxin with cyanogen bromide (CNBr). All three main CNBr-generated fragments of TSST-1, with estimated molecular weights of 20,000, 18,000, and 15,000, reacted with monoclonal antibodies (MAbs) 4T, 5T, 6T, 7T, 8T, and 9T, as determined by autoradiography. The epitopes involved in the mitogenic active site were located on the 15,000-Da internal fragment as determined by the neutralization of the mitogenic activity by the MAb. Modification of the methionine residues in the native TSST-1 by alkylation with iodoacetic acid had no effect on the serologic or mitogenic activity.

Molecular topography of toxic shock syndrome toxin 1 as revealed by spectroscopic studies.

Molecular characterization of toxic shock syndrome toxin 1 has been carried out and compared with a group of functionally related staphylococcal enterotoxins. The secondary structure analysis of the far-UV circular dichroic spectrum of toxic shock syndrome toxin 1 revealed 6.25% alpha-helix, 51.25% beta-pleated sheets, 9.0% beta-turns, and 33.5% random coils. The pattern, in general, was similar to the staphylococcal enterotoxins. Four antigenic sites have been predicted for toxic shock syndrome toxin 1 by using the secondary structure information in combination with the hydrophilicity calculation. The location of the antigenic sites, in general, agrees with the experimental results. Topographical analysis of the tyrosine residues as determined by second-derivative UV spectroscopy [Ragone, R., Colonna, G., Balestrieri, C., Servillo, L., & Irace, G. (1984) Biochemistry 23, 1871-1875] showed that six of nine tyrosine residues are exposed to aqueous solvent. Tryptophan fluorescence quenching studies with an anionic surface quencher, I-, and a neutral quencher, acrylamide, revealed that almost all of the tryptophan residues are buried in the protein matrix as their accessibility to the surface quencher is very low (17%). Since there are only three tryptophan residues in the amino acid sequence of the toxic shock syndrome toxin 1 and there is a tyrosine residue (Tyr-15, Tyr-115, and Tyr-153) next to each of the tryptophan residues (Trp-14, Trp-116, and Trp-154), it appears the tyrosine residues not exposed to the aqueous solvent are those next to the tryptophan residues. Functional implications of the topography of the tryptophan and tyrosine residues are assessed.