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Two studies evaluated growth promoting effects of implant pellets (IP), each containing 3.5 mg estradiol benzoate (EB) and 25 mg trenbolone acetate (TBA), to which a polymeric, porous coating was applied. Trial 1 evaluated performance of heifers (n= 70/treatment, initial BW = 188 ± 2.2 kg) and steers (n= 70/treatment, initial BW = 194 ± 2.2 kg) implanted subcutaneously in the ear with 0 (SC), 2 (2IP), 4 (4IP), or 6 (6IP) pellets that delivered EB/TBA (mg/mg) doses of 0/0, 7/50, 14/100, and 21/150, respectively, over grazing periods of 202 d (heifers) or 203 d (steers). Animals received experimental treatments on d 0 and over the grazing period were managed as single groups by sex in a rotational grazing system. When pasture forage availability became limited, cattle were supplemented with preserved forage but not concentrate supplements. Weight gains by heifers treated with 2IP, 4IP, and 6IP were greater (P< 0.05) than SC heifers but not different from each other. Weight gains by steers treated with 2IP, 4IP, and 6IP were greater than SC steers (P< 0.05), and ADG by steers treated with 6IP was greater (P< 0.05) than steers given 2IP or 4IP. Trial 2 was a multisite grazing study performed with heifers and steers to compare ADG after treatment with one 6-pellet, coated implant delivering 21 mg EB and 150 mg TBA (6IP) to sham treated negative controls (SC) over a grazing period of at least 200 d. A completely random design was used at each site, with the goal to treat 70 cattle per site, treatment, and sex; data were pooled across sites. Heifers (n= 558, initial BW = 229 ± 16 kg) and steers (n= 555, initial BW = 235 ± 20 kg) grazed in rotational programs consistent with regional practices for an average of 202 d. When necessary, cattle were supplemented with preserved forage, but no concentrate supplements were fed. Over 202 d, ADG by heifers treated with 6IP was 11.3% greater (P= 0.0035) than SC heifers (0.64 ± 0.06 kg/d), and ADG by steers treated with 6IP was 17.2% greater (P= 0.0054) than SC steers (0.66 ± 0.08 kg/d). In neither study was there evidence that concurrent therapeutic treatments or abnormal health observations were influenced by experimental treatments. These studies demonstrated that a 6-pellet implant with a polymeric, porous coating that delivers 21 mg EB and 150 mg TBA improved ADG by grazing heifers and steers for at least 200 d compared to sham-implanted negative controls.

Ergovaline has been proposed as a toxic component of endophyte-infected tall fescue. As many of the symptoms of fescue toxicosis are a result of compromised circulation, the objective of this study was to examine the vasoconstrictive potentials of ergovaline and a more documented ergopeptine, ergotamine, using a bovine, lateral (cranial branch) saphenous vein bioassay. Segments of the cranial branch of the lateral saphenous vein (2 to 3 cm) were collected from healthy, mixed breed cattle (n = 12 and n = 5 for the ergovaline and ergotamine experiments, respectively) at local abattoirs. The veins were trimmed of excess fat and connective tissue, sliced into 2- to 3-mm cross sections, and suspended in a myograph chamber containing 5 mL of a modified Krebs-Henseleit, oxygenated buffer (95% O2+ 5% CO2; pH = 7.4; 37°C). The tissue was allowed to equilibrate at 1 g of tension for 90 min before of the addition of treatments. Increasing doses of ergovaline (1×10−11to 1 ×10−4M) or ergotamine (1 ×10−11to 1 × 10−5M) were administered every 15 min after buffer replacement. Contractile response data were normalized to a percentage induced by a reference dose of norepinephrine (1 ×10−4M). Contractile responses of saphenous veins were similar for ergovaline and ergotamine. Initial contractile responses began at 1 ×10−8Mfor both ergovaline and ergotamine (4.4 ± 0.8% and 5.6 ±1.1%, respectively). Vascular tension continued to increase as the alkaloid concentrations increased (maximums: 43.7 ±7.1% at 1 ×10−5Mergotamine; 69.6 ± 5.3% at 1 ×10−4Mergovaline). Interestingly, ergovaline-induced contractions (1 ×10−4M) were not reversed by repeated buffer replacement over a 105-min period. As previously shown with ergotamine, these results confirm that ergovaline is a potent vasoconstrictor. The resistance of an ergovaline-induced contraction to relaxation over an extended period of time suggests a potential for bioaccumulation of this ergopeptine alkaloid and may aid in understanding its toxicity within the animal.

Vasoconstriction has been associated with several symptoms of fescue toxicosis thought to be alkaloid induced. Lysergic acid, an ergot alkaloid, has been proposed as a toxic component of endophyte-infected tall fescue. The objective of this study was to examine the vasoconstrictive potential of D-lysergic acid using a bovine lateral (cranial branch) saphenous vein bioassay. Before testing lysergic acid, validation of the bovine lateral saphenous vein bioassay for use with a multimyograph apparatus was conducted using a dose-response to norepinephrine to evaluate the effects of limb of origin (right vs. left) and overnight storage on vessel contractile response. Segments (2 to 3 cm) of the cranial branch of the lateral saphenous vein were collected from healthy mixed breed cattle (n = 12 and n = 7 for the lysergic acid and norepinephrine experiments, respectively) at local abattoirs. Tissue was placed in modified Krebs-Henseleit, oxygenated buffer and kept on ice or stored at 2 to 8°C until used. Veins were trimmed of excess fat and connective tissue, sliced into 2- to 3-mm sections, and suspended in a myograph chamber containing 5 mL of oxygenated Krebs-Henseleit buffer (95% O2, 5% CO2; pH = 7.4; 37°C). Tissue was allowed to equilibrate at 1 g of tension for 90 min before initiation of treatment additions. Increasing doses of norepinephrine (1 × 10−8to 5 × 10−4M) or lysergic acid (1 × 10−11to 1 × 10−4M) were administered every 15 min after buffer replacement. Data were normalized as a percentage of the contractile response induced by a reference dose of norepinephrine. Veins from both left and right limbs demonstrated contractions in a dose-dependent manner (P< 0.01) but did not differ between limbs. There were no differences in dose-response to norepinephrine between tissue tested the day of dissection and tissue tested 24 h later. Exposure of vein segments to increasing concentrations of lysergic acid did not result in an appreciable contractile response until the addition of 1 × 10−4Mlysergic acid (15.6 ± 2.3% of the 1 × 10−4Mnorepinephrine response). These data indicate that only highly elevated concentrations of lysergic acid result in vasoconstriction. Thus, in relation to the symptoms associated with vasoconstriction, lysergic acid may only play a minor role in the manifestation of fescue toxicosis.

One fifth of 527 cases of hepatitis A occurred in self-identified injection drug users during a community-wide epidemic in Spokane County (Washington) in 1997–8. We hypothesized that an immunization campaign targeted at illicit drug users could control the epidemic. Starting in May 1998, hepatitis A vaccine was provided to individuals in jails and other sites frequented by illicit drug users. Volunteers at vaccination sites were surveyed about risk. Serial convenience samples of jail inmates who denied previous vaccination were anonymously tested for hepatitis A virus (HAV) immunoglobulin G (IgG). From May to December 1998, 2765 high-risk individuals were vaccinated against hepatitis A. The proportion of HAV IgG seropositive inmates increased from 30% to more than 50%. Our findings suggest that vaccination along with naturally occurring infection increased the rate of hepatitis A immunity among illicit drug users during the final months of the epidemic. This supports the hypothesis that targeted immunization of high risk groups may shorten the natural history of a community-wide epidemic.

One fifth of 527 cases of hepatitis A occurred in self-identified injection drug users during a community-wide epidemic in Spokane County (Washington) in 1997–8. We hypothesized that an immunization campaign targeted at illicit drug users could control the epidemic. Starting in May 1998, hepatitis A vaccine was provided to individuals in jails and other sites frequented by illicit drug users. Volunteers at vaccination sites were surveyed about risk. Serial convenience samples of jail inmates who denied previous vaccination were anonymously tested for hepatitis A virus (HAV) immunoglobulin G (IgG). From May to December 1998, 2765 high-risk individuals were vaccinated against hepatitis A. The proportion of HAV IgG seropositive inmates increased from 30% to more than 50%. Our findings suggest that vaccination along with naturally occurring infection increased the rate of hepatitis A immunity among illicit drug users during the final months of the epidemic. This supports the hypothesis that targeted immunization of high risk groups may shorten the natural history of a community-wide epidemic.

It is now generally accepted that activation of tamoxifen occurs as a result of metabolism to alpha-hydroxytamoxifen. In this study, alpha-hydroxytamoxifen was given to female Wistar/Han rats (0.103 or 0.0103 mmol/kg, intraperitoneally, daily for 5 days). This resulted in liver DNA damage, determined by (32)P-post-labelling, of 3333 +/- 795 or 343 +/- 68 adducts/10(8) nucleotides, respectively (mean +/- SD, n = 4). Following HPLC separation, the retention times of the major alpha-hydroxytamoxifen DNA adducts were similar to those seen following the administration of tamoxifen. However, after rats were treated with alpha-hydroxytamoxifen (0.103 mmol/kg) for 5 days and the animals kept for up to 13 months, no liver tumours developed (0/7 rats), even with phenobarbital promotion (0/5 rats). GST-P foci were detected in the liver, but only after 13 months was their number or area significantly increased over the corresponding controls. When alpha-hydroxytamoxifen was given to female lambda/lacI transgenic rats (0.103 mmol/kg orally for 10 days) and the animals killed 46 days later, there was an approximate 1.8-fold increase in mutation frequency but no significant increase in G:C to T:A transversions as described after tamoxifen treatment. It is concluded that DNA damage alone, resulting from the short-term administration of alpha-hydroxytamoxifen, is not sufficient to initiate liver tumours even with phenobarbital promotion. As with tamoxifen, long-term exposure may be required to allow promotion and progression of transformed cells.

Tamoxifen, a rat liver carcinogen, can induce mutations in the lacI gene in the livers of lambda/lacI transgenic rats. However, the presence of persistent tamoxifen adducts on the liver DNA raises the possibility that some contribution to the mutagenesis from ex vivo mutations during the in vitro lacI assay cannot be ruled out. To address this issue, mutagenesis at the cII gene of the transgenic shuttle vector was determined using a selection based assay which is unaffected by the presence of tamoxifen-DNA adducts. Female lambda/lacI transgenic rats were dosed orally with tamoxifen (20 mg/kg body wt) daily for 6 weeks, causing a 3.2-fold increase in the mutant frequency (MF) in the cII gene compared with that obtained with solvent treated animals. This was similar to the MF found previously at the lacI gene and confirms that tamoxifen is mutagenic in vivo. The major class of mutation induced by tamoxifen in the cII gene was G:C-->T:A transversions as was found previously in the lacI gene. However, in the one unreplicated study of mutations in the p53 gene of liver tumours induced by tamoxifen, no G:C-->T:A transversions were found; possible differences between mutagenesis in normal and tumour tissues are explored. The major proportion of the G:C-->T:A transversions occurred at 5'-CpG-3' dinucleotide (CpG) sites in the lacI gene, but not at such sites in the cII gene. The methylation of CpG sites greatly enhances the targeting of deoxyguanosine by carcinogens, thus this finding might be explained by differences in the methylation patterns at their respective CpG sites; however, nothing is known about the methylation status of either the lacI nor the cII gene in this transgenic rat. This study raises the important issue of which target genes (mammalian or transgenic) should be used as endpoints in mammalian mutagenesis assays.

The synthetic β-triketones are a novel family of chemicals, developed as herbicides that have activity on grass and broadleaf weeds and are selective in corn. Toxicological evaluation of a number of these chemicals has established that they interfere with rat hepatic tyrosine catabolism in vivo by inhibiting the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD). This paper describes the kinetics of inhibition of rat hepatic HPPD in vitro by the representative β-triketone 2-[2-nitro-4-(trifluoromethyl)benzoyl]-4,4,6,6-tetramethylcyclohexane-1,3,5-trione (1). A marked inhibition of rat hepatic HPPD was observed when 1 was incubated with the enzyme for 3 min at 37 °C prior to the initiation of the enzyme reaction by the addition of substrate. In this system, a concentration of 200 nM 1 resulted in a >90% loss of HPPD activity, and an apparent IC50 was established at approximately 50 nM. The rate constant for the inactivation of HPPD by 1 was (1.5 ± 0.2) × 10^-5 s^-1 nM^-1 as determined by progress curve data of oxygen consumed by HPPD with time. This inhibition is reversible in that the enzyme−inhibitor complex slowly dissociates, with approximately 5.5 ± 0.6% of the enzyme activity being recovered by 6 h at 25 °C (t1/2, 25 °C, estimated at 101 ± 14 h). In short, our studies establish 1 to be a tight-binding inhibitor of rat hepatic HPPD in vitro. This inhibition is characterized by the rapid inactivation of HPPD by the formation of an enzyme−inhibitor complex that dissociates extremely slowly with recovery of enzyme activity.

We have previously shown that in a murine pre-B lymphocyte cell line, 70Z/3, interleukin-1-induced IgM expression (differentiation) was associated with Na+/H+-mediated cytoplasmic alkalinization. Because interferon-gamma also induces 70Z/3 differentiation, in this study we examined the effects of recombinant murine interferon-gamma (rIFN-gamma) on cell pH. We found that rIFN-gamma (50 units/ml) induced a sustained increase in cell pH, averaging 0.09 pH units above the base line at 30 min and 0.08 at 4 h. Because rIFN-gamma also induced increases in cell Na+ concentration, the data suggests stimulation of Na+/H+ exchange across the cell membrane. Amiloride inhibited the rIFN-gamma-mediated pH rise by only 31% at 30 min, but at 4 h the inhibition was more complete (89%). In contrast to pHi, amiloride totally blocked the Na+ rise at both 30 min and 4 h. This indicates that at the earlier time point the pH rise had a Na+/H+ exchange-dependent and -independent component while at the later time most of the pH rise was due to the Na+/H+ exchanger. The presence of a Na+ independent amiloride-insensitive component was further confirmed by the 0.05 pH rise induced by rIFN-gamma in Na+-free media. Failure to block the Na+-independent rIFN-gamma-mediated pHi rise by anion exchange inhibitors or removal of Ca2+ indicate that this component was not mediated by Cl-/OH- or Ca2+/H+ exchange. The nature of the Na+-independent cell alkalinization process or its role in rIFN-gamma-mediated 70Z/3 differentiation remains to be determined. Because amiloride did not have an effect on rIFN-gamma-induced surface Ig expression or the rIFN-gamma-mediated increase in new kappa light chain-specific mRNA the results indicate that rIFN-gamma-stimulated Na+/H+ is not required for 70Z/3 differentiation.

We have previously shown that recombinant murine interferon-gamma, rIFN-gamma, and recombinant human interleukin-1 alpha, rIL-1 alpha, induce differentiation of murine pre-B-like cell line 70Z/3, a finding associated with stimulation of Na+/H+ exchange across the plasma membrane. The present study was designed to test whether the enhanced Na+/H+ exchange is mediated by Ca2+/phospholipid-dependent protein kinase C. The results show that two structurally different peptides, rIFN-gamma and rIL-1 alpha, induce identical patterns of transient translocation of protein kinase C from the cytosol to the membranes. The increase in membrane-associated protein kinase C activity was first detected 20 min after exposure to the lymphokines. This activity peaked at 30 min and was back to baseline by 2 h. At each time point, the increase in membrane-associated protein kinase C activity corresponded to a decrease in the activity of protein kinase C in the cytoplasmic fraction. The total cellular activity (cytosol + membrane) remained the same. Two series of experiments were carried out to test the role of protein kinase C in mediating the lymphokine-stimulated Na+/H+ exchange. In the first, the effects of rIFN-gamma and rIL-1 alpha on cytoplasmic pH were measured in the presence of a protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, H-7. In the second, rIFN-gamma- and rIL-1 alpha-induced cytoplasmic alkalinization was determined in cells containing decreased protein kinase C activity. Under both experimental conditions, lymphokine-induced cytoplasmic alkalinization was not attenuated. These results indicate that, although both rIFN-gamma and rIL-1 alpha cause association of protein kinase C with membranes, activation of protein kinase C is not required for rIFN-gamma or rIL-1 alpha to stimulate Na+/H+ exchange across the plasma membrane.

To date, studies on the mechanism of toxicity of pesticides are not yet an integral part of the toxicological evaluation process. However, in recent years mechanistic studies have played an increasing role in the assessment of toxicological hazards to man, and in this paper we have described two examples where an understanding of mechanism has contributed positively to risk assessment or has provided a surer scientific basis for the judgement of whether a potential hazard will be expressed in man. In the first example, an evaluation of the scientific literature leads to the conclusion that hepatic peroxisome proliferation in rats and mice is directly, or indirectly, related to the development of hepatocellular tumours. A wide range of non-mutagenic chemicals elicit peroxisome proliferation in mouse and rat liver, but not the guinea pig or marmoset liver. Using one of the diphenyl ether herbicides, fomesafen, we have shown that isolated hepatocytes from mice and rats, but not those from guinea pigs, marmosets and significantly man, undergo peroxisome proliferation. Therefore, it seems reasonable to conclude that although fomesafen causes peroxisome-related tumours in the mouse, man is neither susceptible nor sensitive to this mechanism. Consequently, we can conclude that fomesafen will not cause liver tumours in humans exposed to this herbicide. The herbicide paraquat, although safe in normal agricultural use, has been responsible for numerous human fatalities, almost exclusively as a result of the intentional ingestion of the concentrated commercial product. In this, the second example, studies on the mechanism of paraquat toxicity have provided a rational scientific basis for the evaluation of putative treatments and provide a framework for the development of novel approaches to reduce its toxicity. Also, a study of its mechanism of toxicity revealed that paraquat was selectively accumulated into the lung (in comparison with other tissues) by a previously unknown polyamine transport process. It was possible to determine the apparent kinetic constants for this uptake and use these pharmacokinetic parameters to predict that spray operators exposed to paraquat would not be at risk of lung injury. This scientific argument reinforced the epidemiological evidence that spray operators were clinically indistinguishable from an appropriate control population. In conclusion, mechanistic studies can contribute to the weight of evidence that is required in order to make scientific judgements on the hazards and risks that an individual pesticide will pose to man.

We have reviewed some of the factors which contribute to lung damage by various toxicants. These include disposition of the chemical, its metabolism, individual cell type susceptibility and the potential for the tissue to repair. We have discussed the use of biochemical parameters to measure the functional activity of individual cell types in order to predict the damage to specific cell types and concluded that careful morphological analysis of lung tissue is likely to provide a more sensitive and informative measure of specific cell type injury. However, in order to investigate the mechanism of toxicity of pulmonary toxicants it is essential to establish the primary biochemical event that leads to cell damage and morphological change. The importance of separating the relevant biochemical change(s) from the cascade of biochemical events associated with dead and dying cells and the reparative response of the lung is emphasised.

Tamoxifen was administered to three strains of female mice (B6C3F1, C57BL/6 and DBA/2) in short- and long-term studies to determine their ability to activate tamoxifen and cause hepatic DNA damage. 32P-Postlabelling of liver DNA from mice treated for 4 days showed a group of major adducts that increased in a dose-dependent manner and co-chromatographed with the major adducts detected in rat liver. On cessation of dosing, the majority of adducts were cleared within 3 days. Binding of [14C]tamoxifen to DNA nucleotides was demonstrated by the use of accelerator mass spectrometry. In long-term studies of 12 months to 2 years duration, dependent on strain, tamoxifen was administered continuously in the diet to give a daily dose of approximately 40 mg/kg. DNA adducts were detected after 3 months, although the number of adducts decreased with time and by 2 years were not detectable in the tamoxifen treated mice. None of the treated groups showed a significantly increased incidence of liver tumours, with or without phenobarbital promotion and there was no sustained liver cell proliferation. Tamoxifen was detected in the mouse livers, but at levels 50 times lower than those reported in a comparable rat study. These results suggest that, in contrast to the rat, tamoxifen is non-carcinogenic in mice because it does not cause sufficient cumulative DNA damage, or act as a promoter by causing cell proliferation.

Tamoxifen and its analogues 4-hydroxytamoxifen, toremifene, 4-hydroxytoremifene, clomifene and droloxifene were tested for clastogenic effects in a human lymphoblastoid cell line (MCL-5) expressing elevated native CYP1A1 and containing transfected CYP1A2, CYP2A6, CYP2E1 and CYP3A4 and epoxide hydrolase and in a cell line containing only the viral vector (Ho1). MCL-5 or Ho1 cells were incubated with 4-hydroxytamoxifen, 4-hydroxytoremifene, clomifene or droloxifene and the incidence of micronuclei estimated. With MCL-5 cells there was an increase in micronuclei with 4-hydroxytamoxifen, 4-hydroxytoremifene and clomifene but not with droloxifene. With Ho1 cells only 4-hydroxytamoxifen and 4-hydroxytoremifene caused an increase in micronuclei. MCL-5 cells were incubated with tamoxifen, 4-hydroxytamoxifen, toremifene, droloxifene, clomifene or diethylstilbestrol (0.25-10 microg/ml) for 48 h and subjected to 3 h treatment with vinblastine (0.25 microg/ml) to arrest cells in metaphase. The incidence of cells with chromosomal numerical aberrations (aneuploidy) was increased in cells treated with tamoxifen, 4-hydroxytamoxifen, toremifene, clomifene and diethylstilbestrol but not droloxifene. The frequency of cells with structural abnormalities (excluding gaps) was increased in cells treated with tamoxifen and toremifene but not 4-hydroxytamoxifen, clomifene, droloxifene or diethylstilbestrol. The clastogenic activities of tamoxifen (35 mg/kg), toremifene (36.3 mg/kg), droloxifene (35.2 mg/kg) and diethylstilbestrol (25 mg/kg) were compared in groups of four female Wistar rats. Each chemical was dissolved in glycerol formal, administered as a single dose by gavage and hepatocytes isolated by collagenase perfusion 24 h later. The cells were cultured in the presence of epidermal growth factor (40 ng/ml) for 48 h, colchicine (10 microg/ml) being added for the final 3 h of incubation. At least 100 chromosomal spreads were examined from each animal for the presence of numerical and structural abnormalities. The incidences of aneuploidy following treatment were: tamoxifen 81%, toremifene 46%, droloxifene 9.6%, diethylstilbestrol 45.7%, vehicle control 5.3%. The incidences of chromosomal structural abnormalities excluding gaps were: tamoxifen 4.3%, toremifene 0.8%, droloxifene 0.5%, diethylstilbestrol 0.8%, control 0.5%. The incidence of chromosomal structural aberrations excluding gaps in the treated animals was not statistically significantly different from controls except in the tamoxifen-treated group. Tamoxifen (35 mg/kg per os) and toremifene (36.3 mg/kg per os) were dosed to rats for 4 weeks and chromosomal spreads made from hepatocytes. The incidences of aneuploidy were: tamoxifen 94%, toremifene 57%, control 6.5%. The incidences of chromosomal aberrations excluding gaps were: tamoxifen 12%, toremifene 1%, control 0.5%. The incidence of tamoxifen-induced chromosomal structural abnormalities was significantly elevated compared with control levels. The results demonstrate that tamoxifen and toremifene are the only two drugs tested in the study that cause chromosomal structural and numerical aberrations in vitro and tamoxifen is the only drug that induces both these effects in rat liver cells stimulated to divide in culture following oral dosing. Since chromosomal mutations require cell division for their manifestation and tamoxifen is the only compound of those tested that causes hyperplasia in the rat liver, chromosomal aberrations and aneuploidy in the rat liver would only be expected to occur following treatment with tamoxifen alone, although aneuploidy could be induced by toremifene in conjunction with a promoter such as phenobarbitone.

The purpose of this study was to determine if there were any beneficial or detrimental effects regarding delayed onset muscle soreness (DOMS), serum creatine kinase (CK), and maximum concentric strength at 80% of 1-RMconc, if a bout of eccentric exercise was repeated at 48 h after an initial bout. A secondary purpose was to determine whether unaccustomed eccentrics might affect plasma cholesterol (TC). Twenty-six men were randomly assigned to a control (Group 1) or experimental group (Group 2). Both groups performed three sets (12 repetitions per set) of the eccentric phase of a chest press, at 80% of one repetition maximum (1-RMconc); Group 2 repeated this exercise 48 h later. DOMS and CK were measured before, and every 24 h for 8 days after; TC was measured before, and every 24 h for 4 days. Maximum strength during the concentric phase of a chest press (1-RMconc) was measured before and at 48-h intervals after. A repeated measures analysis of variance revealed a significant time effect (P < 0.05) for DOMS, CK and strength, but no significant difference between groups (P < 0.05). An interesting finding was the significant (P < 0.05) reduction in TC at 24, 48 and 72 h, after exercise in both groups, which we hypothesized was associated with cellular repair. From these results we concluded that when a bout of eccentrics is repeated 48 h after an initial bout, there is no change in the characteristic time-course and/or intensity of DOMS, CK or 1-RMconc.

The separation of toremifene and its metabolites 4-hydroxytoremifene, N-desmethyltoremifene, N-desdimethyltoremifene and deaminohydroxytoremifene by reversed-phase high performance liquid chromatography is described. The effects of pH, buffer concentration and type and proportion of organic modifier on the retention and resolution of the compounds have been studied. This allows optimum conditions for a particular biological application to be developed by simple modification of these paramaters. For the separation of toremifene and metabolites in microsomal metabolism and in plasma, the optimum conditions were 65% (v/v) acetonitrile in 0.25M ammonium acetate-acetic acid buffer, pH 5.0-5.2.

We have previously shown that interleukin-la (IL-1α) and interferon-γ (IFN-γ) induce surface IgM expression, stimulate Na+/H+exchange, and activate protein kinase C in the murine pre-B lymphocyte cell line, 70Z/3. Because the two structurally different lymphokines induce similar effects, in this study we set out to compare the properties of the IL-1 and IFN-γ surface receptors. In contrast to their similar cellular effects, we found that IL-1α and IFN-γ receptors have different properties. 70Z/3 have high (100 sites/cell) and low (900 sites/cell) affinity IL-1 receptors with dissociation constants (KD) 6 × 10−11and 10−9M, respectively. In contrast, IFN-γ receptors are of one class with a KDof 3 × 10−10Mand are at a higher number, 8000 sites/cell. After binding to their receptors both IL-1α and IFN-γ are internalized and intracellularly degraded, but the rate of internalization of IFN-γ is greater than IL-1α. The effective median concentrations (EC50) of IL-1α- or IFN-γ-induced surface IgM expression are similar (4–5 × 10−12M). However, at this concentration 10-fold more of IFN-γ than IL-1α molecules are bound per cell.

To investigate the activation mechanisms involved in tamoxifen carcinogenicity, analogues of tamoxifen isomers modified at the ethyl group were synthesized and assessed for their ability to induce hepatic DNA damage following their administration to female F344 rats. The cis isomer was prepared by acid-catalyzed isomerization of tamoxifen and isolated by preparative HPLC. The active metabolite α-hydroxytamoxifen and geometric isomers of bromotamoxifen and C-desmethylenetamoxifen, analogues in which the ethyl group has been replaced by a bromine atom and methyl group, respectively, were synthesized according to published procedures. The levels of hepatic DNA adducts induced were determined by ³²P-postlabeling. Bromotamoxifen and tamoxifen 1,2-epoxide caused no detectable DNA damage relative to controls. Trans isomers of tamoxifen, C-desmethylenetamoxifen, and α-hydroxytamoxifen all produced DNA adducts at a 5−90-fold higher level than the corresponding cis isomers. In contrast, both the cis and trans isomers of α-hydroxytamoxifen showed similar reactivity toward calf thymus DNA in vitro. Molecular models of α-hydroxytamoxifen isomers suggest this difference in DNA adduct-forming ability is due to steric hindrance of the enzymes involved in the activation of this metabolite. There were high adduct levels in the liver, but no uterine DNA adducts were detected in rats treated with α-hydroxytamoxifen. This suggests that in contrast to the liver, α-hydroxytamoxifen is not further activated in rat uterus. This may help to explain the absence of uterine tumors in rats following long-term tamoxifen treatment.

Hb Gun Hill has been found in an asymptomatic black female living in Alabama; neither her parents nor her two siblings carry the variant. The deletion of five mino acid residues in the β chain of lib Gun Hill (α2β2 91-95 deleted) causes this vartant to have a high oxygen affinity, an interaction coefficient of unity and an absence of the Bohr effect. The rate of oxidation of liganded Hb Gun Hill by the ferricyanide ion follovs first-order kinetics. Hb Gun Hill is to a great extent dissociated into dimers over a wide range of pH values.

Dominant commercial treatments currently used for wood preservation are pentachlorophenols (PCPs), creosote, and chromium copper arsenicals (CCAs). These wood preservatives present many studied and documented environmental and toxicological problems both with use of the products themselves and with the production of secondary reaction contaminants. Furthermore, several of these preservatives are considered priority pollutants according to the U.S. Environmental Protection Agency (EPA) and are heavily regulated. The need for an environmentally benign combined fire/rot retardant treatment for wood that will effectively reduce both fire and decay is clearly evident. Guayule resin, a co product during rubber extraction from the guayule shrub, is being explored by us as a natural, biodegradable wood rot and fire retardant with potential commercial applications. Wood impregnated with guayule resin has survived over 8 years in a tropical rain forest environment, free of rot, decay, and insect infestations. This study qualitatively analyzed treated and untreated wood samples For ignitability, flame spread, toxic fumes, and smoke generation. Ignitability of the wood sample of fixed geometry was quantified by measuring the time required for ignition of treated samples versus untreated samples. Flame spread was measured after ignition with a propane flame by a simple stopwatch-ruler technique. Smoke mass was determined in an Arapa-hoe Smoke Chamber as per ASTM D-4100. Analysis of the fumes involved continuous CO, C02, NOx, SOx, and O2 analyses using a Bacharach Instrument Model NSX 300. An Andersen Inc. Model 2000 cascade impactor was used to determine the particlesize distribution of the smoke and for smoke mass. Most of the differences in the test data measurements between guayule-impregnated pine and untreated pine were found to be minor. However, the ignition data indicated that guayule-impregnated pine did not ignite as quickly as untreated pine. Based on the favorable data presented herein, further research of wood preservation with guayule resin is recommended.

Osteopontin is an adhesive glycoprotein implicated in numerous diseases associated with inflammation and remodeling. There are several structural domains in osteopontin that are of particular interest. The RGD motif is a cell attachment sequence shown to be critical for cell adhesion through alphav-containing integrins. In close proximity to the RGD domain is the thrombin cleavage site. Previous observations suggest that thrombin cleavage of osteopontin occurs in vivo and may be physiologically important. To study the functional significance of osteopontin cleavage by thrombin, we made glutathione S-transferase-osteopontin fusion proteins. These proteins contain either the N- or C-terminal domains expected to be formed following thrombin cleavage at the Arg169-Ser170 peptide bond. We compared these osteopontin fragments with native osteopontin in their ability to support adhesion of several different cell lines and identified the receptors mediating these interactions. Our data show that the N-terminal osteopontin fragment, which contains the RGD domain, supports adhesion of a melanoma cell line that is unable to bind native osteopontin. This suggests that osteopontin adhesive interactions may be regulated by thrombin cleavage. We also demonstrate that osteopontin contains a cryptic binding activity, which can be recognized by a novel osteopontin receptor. This receptor has been identified as the alpha9beta1 integrin.

The work described in this paper is part of a systematic study of surface cleaning and ohmic contact strategies for GaN. The goal of this investigation was to determine the most effective methods of wet chemical and thermal desorption cleaning for the removal of oxygen (O) and carbon (C) prior to metallization. Hydrochloric (HC1) and hydrofluoric (HF) acid-based cleaning treatments were compared, and thermal desorption as a function of temperature was characterized by sequential heating under ultra high vacuum (UHV) conditions. Auger electron spectroscopy (AES) analysis was used to monitor the presence of surface O and C throughout the study. For the removal of surface oxide, HCl-based solutions were found to be most effective; under as-cleaned, air-exposed conditions, HC1:DI H2O (1:1) solution resulted in the lowest levels of residual O and C. However, HF-based solutions resulted in more effective thermal desorption of C from the surfaces. In contrast to the results typically observed in the thermal desorption cleaning of GaAs, complete removal of oxygen and carbon from airexposed GaN surfaces was not seen using vacuum heating alone, even to temperatures where GaN decomposition occurs (>800-900°C). The results of this study indicate that the presence of oxygen and carbon on the GaN surface is persistent even to high temperatures, and that further in-situ cleaning methods must be added to obtain spectroscopically clean GaN surfaces.

An inducible expression vector system has been developed to facilitate the study of the effects of individual virus gene products on cell function. The vector utilizes the mouse metallothionein promoter carried on the bovine papillomavirus genome. Conditions which optimize the induced expression of open reading frames inserted downstream from the mouse metallothionein promoter have recently been described. In this communication we describe the use of this system to clone and express the encephalomyocarditis virus 3C protease in cultured mouse cells. Stably transformed cell lines could be induced to produce levels of 3C protease activity comparable to those observed during normal virus infection. In spite of this, no effects on cellular protein synthesis rate or membrane permeability were observed. It was also discovered that 3C protease as well as 3C protease-containing polyproteins are turned over. This was true not only in the induced cell clones, but also during the normal course of encephalomyocarditis virus infection, as well as in translation systems in vitro. This phenomenon was highly specific for this family of polypeptides, perhaps explaining their apparent lack of cytotoxic effects.

A novel HPLC system has been developed that has allowed the separation of tamoxifen DNA adducts formed in the livers of rats and mice treated with this drug. At least 13 different peaks have been separated from 32P-post-labelled DNA, with two major peaks jointly accounting for >60% of the total adducts formed by tamoxifen in the livers of treated rats and mice. This is a great improvement on the resolution obtained by thin layer chromatography, which separates the adducts into one main product consisting of a group of major adduct spots eluting together, plus several other minor spots. Identification of the nature of some of the peaks has been investigated. Comparisons of the products formed when alpha-acetoxytamoxifen is reacted with DNA in vitro with 32P-post-labelled liver DNA adducts from rats treated with tamoxifen or alpha-hydroxytamoxifen in vivo, appear to confirm that a major route of activation of tamoxifen in vivo is via alpha-hydroxylation. The resolving power of this HPLC system has further extended this result to show that six of the peaks, including the two major peaks, are formed by the reaction of an activated alpha-hydroxytamoxifen with DNA. Activation of 4-hydroxytamoxifen by the peroxidase/H2O2 system in vitro gives a more polar DNA adduct seen only at trace levels in liver DNA from tamoxifen-treated rats and mice.

Rats administered tamoxifen for 3 months and then returned to a basal diet developed an increase in uterine weight for up to 9 months after tamoxifen exposure. Stereological analysis of the tamoxifen exposed rat uteri showed that there was a significant increase in the amount of uterine myometrium, for a further 9 months, subsequent to the discontinuation of tamoxifen. A low incidence of myometrial proliferations (deciduomas) and uterine tumours was found at the conclusion of the study (20 months). In contrast, continuous administration of tamoxifen to mice for 24 months produced hyperplasia of the uterine endometrial epithelium and atrophy of the myometrium for the first 3 months, followed by atrophy of both the endometrium and myometrium for the remaining 21 months of the study. No uterine tumours were found in mice treated with tamoxifen for 2 years. The use of stereological analysis on interim sacrifice rodent uteri indicated that sustained uterine tissue compartment effects can occur, with either the continuous administration of tamoxifen, or after its discontinuation. Tamoxifen can have an agonist and antagonist like effect on oestrogen activity in different tissue compartments of the mouse uterus, over the same time period. The particular relevance of the finding of uterine proliferation and atrophy in the rodent studies with tamoxifen is discussed with regard to women taking this drug.

Tamoxifen induces hepatocellular carcinomas in rats and is converted by rat hepatic cytochrome P450 enzymes into reactive metabolites capable of forming adducts with nucleic acids, proteins and chromosomal aberrations. In rats tamoxifen has also been shown to induce liver cytochrome P450 enzymes, to stimulate its own metabolism leading to greater covalent binding and to induce a higher degree of unscheduled DNA synthesis. This suggests that, at least in the rat, a sensitive species, tamoxifen may contribute significantly to its genotoxic and carcinogenic potential, by assisting its own metabolic activation. We have now investigated the effect of feeding tamoxifen to male and female Rhesus monkeys. A marked induction of the hepatic cytochrome(s) P450 is found in the monkey but, in spite of this, the in vitro metabolism of 7-ethoxyresorufin by microsomes from treated animals is markedly inhibited and so is the dealkylation of two other 7-alkoxyresorufin substrates. Evidence is presented for the accumulation in the liver of monkeys treated with tamoxifen of a powerful inhibitor of drug metabolism, and the inhibitor is identified as a metabolite of tamoxifen, its N,N-didesmethyl derivative. The level of 32P-postlabelled DNA adducts was considerably higher in rats given tamoxifen than in similarly treated monkeys. Also, whereas rats responded to tamoxifen treatment with a marked increase in covalent binding to microsomal protein, in the monkeys, where accumulation of the inhibitory metabolite in the microsomal fraction was also seen, covalent binding was not greater with microsomes from treated animals than in the corresponding controls. N,N-Didesmethyl-tamoxifen, added in vitro to human and rat microsomes, reduced significantly the extent of covalent binding, suggesting that the accumulation of the metabolite observed in the liver of primates may discourage the cytochrome P450-dependent conversion of tamoxifen into reactive derivatives and in this way protect against the formation of adducts. This mechanism may also contribute to protecting the primate against tamoxifen- induced liver cancer.

Previous laboratory studies have shown that nitrapyrin (2‐chloro‐6‐(trichloromethyl) pyridine) can produce phytotoxicity in certain plants. Nitrapyrin hydrolyzes in soil to 6‐chloropicolinic acid; however, the phytotoxic properties of the latter compound have not been compared with those of the former. Therefore, studies were conducted to compare the relative toxicity of nitrapyrin and 6‐chloropicolinic acid to the seedlings of various crop plants, to compare their phytotoxicity in a variety of soils, and to estimate if the soil concentration, of either chemical, required to produce significant growth reduction is less than recommended field application rates for nitrapyrin. Nitrapyrin was more toxic than 6‐chloropicolinic acid to the graminaceous species tested which included corn (Zea maysL.), sorghum (Sorghum vulgateL.), wheat (Triticum aestivumL.), and rice (Oryza sativaL.). 6‐Chloropicolinic acid was more toxic than nitrapyrin to the dicotyledenous species tested which included cotton (Gossypium hirsutumL.), sugar beet (Beta vulgarisL.), tomato (Lycopersicon esculentumL.), alfalfa (Medicago sativaL.), and soybean (Glycine maxL.). The dicotyledenous species were more sensitive to both compounds than were the graminaceous species. All species tested except alfalfa and tomato tolerated soil concentrations of nitrapyrin estimated to be greater than the highest recommended field application rates. Nitrapyrin was more highly adsorbed and therefore detoxified to a greater extent than 6‐chloropicolinic acid when soil organic matter increased.

A 17-yr-old black male with hemolysis and pigmenturia but no anemia was found to have hemoglobin Köln (α2β298 val→met [FG5]). Splenectomy was done because of complicating thrombocytopenia. Thrombokinetic studies with 51Cr tagged platelets suggested hypersplenism, and after surgery the platelet count returned to normal. The red cell t ½ 51Cr was more than doubled, but the red cell life span (DF32P) was more modestly improved (30.6 → 47.2 days). The "elution" of 51Cr from the red cells presplenectomy was 5.6%/day, whereas after surgery it was normal (1.9%/day), accounting for the disparity between the survival methods. Study of the isolated cyanferri derivative of hemoglobin Köln by ultracentrifugation at various salt concentrations and various pH’s indicated an increased tendency to dimer formation under conditions where normal hemoglobin is a tetramer. This results from the site and type of amino acid substitution and accounts in part for its instability.

GaN is a semiconductor with a direct band-gap of 3.4 eV which has a potential application in optoelectronic devices such as UV-emitting lasers and blue light emitting diodes. Extensive efforts have been made to develop low-resistance, thermally stable and uniform ohmic contacts on GaN. In this study, n-GaN films (Si-doped) with an intermediate A1N buffer layer were grown on 6H-SiC substrates via MOVPE. The contacts were formed by depositing Ti and then Au films on GaN by e-beam evaporation at room temperature. Current-voltage measurements as a function of temperature showed that the ohmicity of the Au/Ti contact improved upon annealing, corresponding to changes in the microstructure of the contact interfaces involving a redistribution of nitrogen and gold.Fig. 1 shows the microstructure of the as-deposited Au/Ti/GaN films. The nanocrystalline Ti film was about 50 nm thick. Au film was polycrystalline, containing many growth twins. The thickness of Au film varied from 260 to 350 nm.

GaN is a semiconductor with a direct band-gap of 3.4 eV which has a potential application in optoelectronic devices such as UV-emitting lasers and blue light emitting diodes. Extensive efforts have been made to develop low-resistance, thermally stable and uniform ohmic contacts on GaN. In this study, n-GaN films (Si-doped) with an intermediate A1N buffer layer were grown on 6H-SiC substrates via MOVPE. The contacts were formed by depositing Ti and then Au films on GaN by e-beam evaporation at room temperature. Current-voltage measurements as a function of temperature showed that the ohmicity of the Au/Ti contact improved upon annealing, corresponding to changes in the microstructure of the contact interfaces involving a redistribution of nitrogen and gold.Fig. 1 shows the microstructure of the as-deposited Au/Ti/GaN films. The nanocrystalline Ti film was about 50 nm thick. Au film was polycrystalline, containing many growth twins. The thickness of Au film varied from 260 to 350 nm.