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Calf-fed Holstein steers (n = 115; 449 ± 20 kg) were utilized in a serial harvest experiment. A baseline group of five steers was harvested after 226 d on feed (DOF), which was designated day 0. The remaining cattle were assigned randomly to 11 harvest groups, with slaughter every 28 d. Cattle were either not (CON) or were fed zilpaterol hydrochloride for 20 d followed by a 3 d withdrawal (ZH). There were five steers per treatment in each slaughter group ranging from days 28 to 308. Whole carcasses were divided into lean, bone, internal cavity, hide, and fat trim components. Apparent mineral retention (Ca, P, Mg, K, and S) within the body was calculated as the difference between mineral concentration at slaughter and day 0. Mineral concentration at day 0 was determined from body composition of steers harvested at day 0 multiplied by individual live body weight (BW) at day 0. All data were analyzed as a 2 × 11 factorial arrangement with individual animal as the experimental unit. Orthogonal contrasts were used to analyze linear and quadratic contrasts over time (11 slaughter dates). There were no differences in concentration of Ca, P, and Mg in bone tissue as feeding duration increased (P ≥ 0.89); concentration of K, Mg, and S in lean tissue did fluctuate across DOF (P < 0.01). Averaged across treatment and DOF, 99% of Ca, 92% of P, 78% of Mg, and 23% of S present in the body were in bone tissue; 67% of K and 49% of S were in lean tissue. Expressed as gram per day, apparent retention of all minerals decreased linearly across DOF (P < 0.01). Expressed relative to empty body weight (EBW) gain, apparent Ca, P, and K retention decreased linearly as BW increased (P < 0.01) whereas Mg and S increased linearly (P < 0.01). Apparent retention of Ca was greater for CON cattle (greater bone fraction) and apparent retention of K was greater for ZH cattle (greater muscle fraction) when expressed relative to EBW gain (P ≤ 0.02), demonstrating the increase in lean gain by ZH cattle. There were no differences in apparent retention of Ca, P, Mg, K, or S due to treatment (P ≥ 0.14) or time (P ≥ 0.11) when expressed relative to protein gain. Apparent retention averaged 14.4 g Ca, 7.5 g P, 0.45 g Mg, 1.3 g K, and 1.0 g S/100 g protein gain. Expressing apparent mineral retention on a protein gain basis minimized effects of rate and type of gain, allowing for better comparison across treatments and time. Feeding zilpaterol hydrochloride did not affect apparent mineral retention when expressed relative to protein gain., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Heat stress (HS) triggers oxidative stress, systemic inflammation, and disrupts growth efficiency of livestock. β-adrenergic agonists supplemented to ruminant livestock improve growth performance, increase skeletal muscle mass, and decrease carcass fat. The objective of this study was to understand the independent and interacting effects of HS and zilpaterol hydrochloride (ZH) supplementation on the transcriptome of subcutaneous white adipose tissue and the longissimus dorsi muscle in steers. Twenty-four Red Angus-based steers were assigned to thermoneutral (TN; Temperature Humidity Index [THI] = 68) or HS (THI = 73-85) conditions and were not supplemented or supplemented with ZH (8.33 mg/kg/d) for 21 d in a 2 × 2 factorial. Steers in the TN condition were pair-fed to the average daily feed intake of HS steers. RNA was isolated from adipose tissue and skeletal muscle samples collected via biopsy on 3, 10, and 21 d and sequenced using 3' Tag-Seq to an achieved average depth of 3.6 million reads/sample. Transcripts, mapped to ARS-UCD1.2, were quantified. Differential expression (DE) analyses were performed in DESeq2 with a significance threshold for false discovery rate of 0.05. In adipose, 4 loci (MISP3, APOL6, SLC25A4, and S100A12) were DE due to ZH on day 3, and 2 (RRAD, ALB) were DE due to the interaction of HS and ZH on day 10 (Padj < 0.05). In muscle, 40 loci (including TENM4 and OAZ1) were DE due to ZH on day 10, and 6 loci (HIF1A, LOC101903734, PDZD9, HNRNPU, MTUS1, and TMCO6) were DE due to environment on day 21 (Padj < 0.05). To explore biological pathways altered by environment, supplement, and their interaction, loci with DE (Praw < 0.05) were evaluated in Ingenuity Pathway Analysis. In adipose, 509 pathways were predicted to be altered (P < 0.01): 202 due to HS, 126 due to ZH, and 181 due to the interaction; these included inflammatory pathways predicted to be upregulated due to HS but downregulated due to the interaction of HS and ZH. In muscle, 113 pathways were predicted to be altered (P < 0.01): 23 due to HS, 66 due to ZH, and 24 due to the interaction of HS and ZH. Loci and pathway data in muscle suggest HS induced oxidative stress and that the stress response was moderated by ZH. Metabolic pathways were predicted to be altered due to HS, ZH, and their interaction in both tissues. These data provide evidence that HS and ZH interact to alter expression of genes in metabolic and immune function pathways and that ZH moderates some adverse effects of HS., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

An experiment was conducted to evaluate the fabrication yields of carcasses from beef steers supplemented zilpaterol hydrochloride (ZH) and fed at maintenance (MA) or ad libitum (AB) intake levels. Beef steers (n = 56) from a common sire were blocked (n = 28 per block) by terminal growth implant and sorted into pairs by BW. Four pairs (n = 8) were harvested on day 0; the remaining 24 pairs (n = 48) were assigned to a dietary intake level (MA or AB) and days on feed (28 or 56 d). Within pairs of MA or AB intakes, steers harvested on day 56 were randomly assigned to supplementation of ZH (90 mg·d-1 per steer) for 20 d followed by a withdrawal period of 4 d or control (C). Steers (BW = 603.5 ± 48.1 kg) were harvested at a commercial processing facility. After a 24-h chill period, standard USDA grading procedures were used to derive a calculated yield grade and quality grade. Following grading, left carcass sides were transported to the West Texas A&M University Meat Laboratory for fabrication. Each side was fabricated into subprimals to determine individual red meat yield (RMY), trimmable fat yield (TFY), and bone yield (BY). A mixed model was used for analysis; fixed effects included treatment combinations and random effects included block and pairs. Single df contrasts tested day 0 vs. 28, day 0 vs. 56, day 28 vs. 56, MA vs. AB, and C vs. ZH. Yield of chuck eye roll differed (P = 0.05) by days on feed (0 d = 4.14, 28 d = 4.11, 56 d = 4.55%). Similarly, eye of round yield was impacted (P = 0.02) by days on feed (0 d = 1.51, 28 d = 1.37, 56 d = 1.36%). Additionally, brisket yield was altered (P < 0.01) by days on feed (0 d = 4.08, 28 d = 3.56, 56 d = 3.48%) and treatment (C = 3.34, ZH = 3.61%). For remaining subprimals, no differences (P ≥ 0.15) were detected. Furthermore, results indicated that RMY tended (P = 0.07) to differ by treatment (C = 61.35, ZH = 63.67%). Comparatively, TFY was impacted (P = 0.04) by intake (MA = 20.44, AB = 23.33%). Results from this study indicate that a MA intake level during the last 56 d of the finishing period concurrent with ZH supplementation impacts subprimal yields as well as carcass RMY and TFY of beef steers.

A trial was conducted to examine live growth efficiency, harvest yields, and carcass grading performance of steers fed at maintenance (M) or at ad libitum (A) level of intake during zilpaterol hydrochloride (Z) supplementation. Single-sired, beef steers (n = 56; start of trial BW 590 ± 36 kg) blocked (n = 2) by weight and terminal implant were sorted into pairs (n = 14 per block) by weight. Pairs of steers were initially assigned to 0, 28, or 56 d of feeding. Within 28 or 56 d, pairs were assigned to M or A intake. Steers within a pair assigned to 56 d of feeding were randomly assigned to either 20 d of Z supplementation (90 mg/d per steer) with a 4 d withdrawal period prior to slaughter or to no ZH supplementation (C). Steers were housed and fed in individual pens. Weights of all non-carcass and carcass components were recorded at slaughter; carcasses were graded 24-h postmortem. Data were analyzed via a mixed model; the fixed effect was treatment combination with random effects of block and pair. Live growth data used harvest day as the repeated measure and animal as the subject. Single df contrasts were constructed for day 0 vs. day 28, day 0 vs. day 56, day 28 vs. day 56, M vs. A, and C vs. Z. Treatment impacted (P ≤ 0.05) live ADG; contrasts indicated A (1.33) was greater than M (0.14 kg), and Z (1.12) was greater than C (0.82 kg). Similarly, carcass ADG differences (P < 0.01) indicated A (1.04) was greater than M (0.36 kg), and Z (1.35) was greater than C (0.71 kg). Intake level altered BW and empty body weight (EBW); M cattle had reduced BW and EBW (P < 0.01, 585 and 540 kg) than A cattle (647 and 597 kg). Cattle fed at M had less carcass and internal cavity mass (P < 0.01, 359 and 79.4 kg) than A cattle (394 and 93.5 kg). Liver mass was reduced by M feeding (P < 0.01; M-5.03, A-6.69 kg) and Z treatment (P < 0.01; Z-5.64, C-6.06 kg). Moreover, mass of total splanchnic tissue was less (P < 0.01) for M cattle than A cattle (59.8 vs. 72.5 kg). Dressed carcass yield was greater (P < 0.01) for Z than C cattle (63.5 vs. 61.6%). Cattle fed at M had less 12th rib s.c. fat, lower numerical U.S. yield grades (P < 0.01; M-1.71 cm and 3.3, A-2.46 cm and 4.3) and lower numerical Canadian yield grades (P < 0.01; 51.9 vs. 53.9% for M and A, respectively) than A cattle. Results indicate that energy intake level and Z supplementation influence live and carcass growth, carcass transfer, kill yields, and carcass characteristics across time.

Interactive effects of supplemental Zn and zilpaterol hydrochloride (ZH) were evaluated in feedlot steers ( = 40; 652 kg ± 14 initial BW) to determine their impact on feedlot performance, blood constituents, and carcass traits. The study was conducted as a randomized complete block design with a 2 × 2 factorial treatment arrangement. Steers were blocked by BW and randomly assigned to treatments. Factors consisted of supplemental Zn (60 or 300 mg/kg diet DM) and ZH (0 or 8.33 mg/kg) in the diets. For diets supplemented with 300 mg Zn/kg DM, 60 mg Zn/kg was supplemented as zinc sulfate and 240 mg Zn/kg was supplemented as zinc oxide, and the diet was fed for 24 d. Zilpaterol hydrochloride was fed for 21 d followed by a 3-d withdrawal. Cattle were housed in partially covered individual feeding pens equipped with automatic waterers and fence-line feed bunks and were fed once daily for ad libitum intake. Plasma samples were collected on d 0 and 21 to assess changes in Zn, plasma urea nitrogen (PUN), glucose, and lactate concentrations, and serum samples were collected on d 21 to assess IGF-1 concentration. On d 25, cattle were weighed and transported 450 km to a commercial abattoir for harvest; HCW and incidence of liver abscesses were recorded. Carcass data were collected after 36 h of refrigeration. Data were analyzed as a mixed model with Zn, ZH, and Zn × ZH as fixed effects; block as a random effect; and steer as the experimental unit. No interaction or effects of Zn or ZH were observed for IGF-1 concentration, plasma glucose, or lactate concentrations ( ≥ 0.25). No interaction between Zn and ZH was observed for PUN concentration, but PUN decreased with ZH ( < 0.01). There were no effects of ZH or Zn on ADG, DMI, final BW, feed efficiency, HCW, back fat, KPH, quality grade, or incidence of liver abscesses ( > 0.05). Zinc supplementation tended ( = 0.08) to improve the proportion of carcasses grading USDA Choice. Feeding ZH decreased yield grade ( = 0.05) and tended to increase LM area ( = 0.07). In conclusion, increasing dietary concentrations of Zn does not impact response to ZH, but feeding ZH altered circulating concentrations of PUN.

Recent studies show that adrenergic agonists and inflammatory cytokines can stimulate skeletal muscle glucose uptake, but it is unclear if glucose oxidation is similarly increased. Thus, the objective of this study was to determine the effects of ractopamine HCl (β1 agonist), zilpaterol HCl (β2 agonist), TNFα, and IL-6 on glucose uptake and oxidation rates in unstimulated and insulin-stimulated soleus muscle strips from adult Sprague-Dawley rats. Effects on phosphorylation of Akt (phospho-Akt), p38 MAPK (phospho-p38), and p44/42 MAPK (phospho-p44/42) was also determined. Incubation with insulin increased (P<0.05) glucose uptake by ∼47%, glucose oxidation by ∼32%, and phospho-Akt by ∼238%. Insulin also increased (P<0.05) phospho-p38, but only after 2h in incubation. Muscle incubated with β2 agonist alone exhibited ∼20% less (P<0.05) glucose uptake but ∼32% greater (P<0.05) glucose oxidation than unstimulated muscle. Moreover, co-incubation with insulin+β2 agonist increased (P<0.05) glucose oxidation and phospho-Akt compared to insulin alone. Conversely, β1 agonist did not appear to affect basal or insulin-stimulated glucose metabolism, and neither β agonist affected phospho-p44/42. TNFα and IL-6 increased (P<0.05) glucose oxidation by ∼23% and ∼33%, respectively, in the absence of insulin. This coincided with increased (P<0.05) phospho-p38 and phospho-p44/42 but not phospho-Akt. Furthermore, co-incubation of muscle with insulin+either cytokine yielded glucose oxidation rates that were similar to insulin alone, despite lower (P<0.05) phospho-Akt. Importantly, cytokine-mediated increases in glucose oxidation rates were not concomitant with greater glucose uptake. These results show that acute β2 adrenergic stimulation, but not β1 stimulation, directly increases fractional glucose oxidation in the absence of insulin and synergistically increases glucose oxidation when combined with insulin. The cytokines, TNFα and IL-6, likewise directly increased glucose oxidation in the absence of insulin, but were not additive in combination with insulin and in fact appeared to disrupt Akt-mediated insulin signaling. Rather, cytokines appear to be acting through MAPKs to elicit effects on glucose oxidation. Regardless, stimulation of glucose oxidation by these key stress factors did not rely upon greater glucose uptake, which may promote metabolic efficiency during acute stress by increasing fractional glucose oxidation without increasing total glucose consumption by muscle., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

An indirect calorimetry experiment was conducted to determine the effects of feeding zilpaterol hydrochloride (ZH) for 20 d on total body oxygen consumption, respiratory quotient, methane production, and blood metabolites in finishing beef steers. Sixteen Angus steers (initial BW = 555 ± 12.7 kg) were individually fed at ad libitum intake and used in a completely randomized design. The model included the fixed effects of dietary treatment, day, and treatment × day. Dry matter intake did not differ between the treatments ( = 0.89), but was greater on d 0 than any other day ( < 0.01). Oxygen consumption was not different between treatments ( = 0.79), but was different across day ( < 0.01) on d 7, 14, 21, and 28. Respiratory quotient was less for cattle fed ZH than control ( < 0.01), and also different across day ( < 0.01), being greater on d 7, 21, and 28 than d 3 or 21. Methane production (L/kg of DMI) was greater for steers fed the control vs. the ZH diet ( < 0.01), and it also differed by day ( < 0.01), being greater on d 21 and 28 than d 0, 3, 7, and 14. Nonesterified fatty acids were not different across treatments ( = 0.82), and there was no effect of treatment on β-hydroxybutyrate concentration ( = 0.45). Whole blood glucose concentrations were not affected by feeding ZH in this experiment ( = 0.76); however, lactate concentrations were reduced by feeding ZH ( = 0.03). Additionally, there was no treatment effect on ɑ-amino-N, blood glutamate, or glutamine ( ≥ 0.16). Plasma NH was not affected by ZH ( = 0.07), but plasma urea nitrogen was reduced by ZH ( < 0.01). Urinary creatinine was increased by steers receiving ZH ( = 0.01), and urine 3-methylhistidine (3-MH) concentrations were normalized to creatinine, the 3-MH:creatinine ratio decreased from d 0 to d 3 in steers fed ZH, and remained less than control steers until d 28. These data provide insight into how β-agonists alter nutrient partitioning and improve the efficiency of tissue accretion, mainly through decreased muscle protein turnover and altering the catabolic fuel for peripheral tissues.

Holstein steers ( = 110) were fed zilpaterol hydrochloride (ZH) for 0 or 20 d before slaughter during a 280-d serial harvest study. Cattle were harvested every 28 d beginning at 254 d on feed (DOF) and concluding at 534 DOF. After slaughter, carcasses were chilled for 48 h and then fabricated into boneless closely trimmed or denuded subprimals, lean trim, trimmable fat, and bone. Inclusion of ZH increased cold side weight (CSW) by 10.3 kg ( < 0.01; 212.7 vs. 202.4 kg [SEM 1.96]) and saleable yield by 10.4 kg ( < 0.01; 131.9 vs. 121.5 kg [SEM 1.16]) in calf-fed Holstein steer carcasses. Additionally, saleable yield as a percentage of CSW increased ( ≤ 0.01) by 2.19% (62.64 vs. 60.45% [SEM 0.22]) for cattle supplemented with ZH. Subprimal weights were heavier ( ≤ 0.05) from cattle that received ZH except for the bottom sirloin ball tip, back ribs, and outside skirt regardless of slaughter endpoint. Yield of top round, bottom round, and knuckle was increased ( ≤ 0.01) following ZH supplementation by 0.37, 0.24, and 0.18%, respectively. Yield of the top sirloin butt, strip loin, and tenderloin was increased ( ≤ 0.01) concurrent with ZH supplementation by 0.18, 0.11, and 0.09%, respectively. Regarding the rib primal, the rib eye roll tended ( = 0.08) to had increased yield (2.80 vs. 2.72% [SEM 0.03]) with ZH supplementation; both back ribs and blade meat exhibited increased ( ≤ 0.04) yields of 0.04%. Relative to the chuck primal, increased ( ≤ 0.03) yields of shoulder clod, pectoral meat, and mock tender were observed (0.13, 0.07, and 0.04%, respectively). Yield changes for subprimal brisket, plate, and flank were limited to increased ( < 0.01) proportion of flank steak and elephant ear (cutaneous trunci), 0.07 and 0.04%, respectively. Feeding duration notably altered ( ≤ 0.01) weights and percentages of all subprimals except the brisket. Saleable yield increased ( ≤ 0.01) by 0.192 kg/d with additional DOF. Moreover, trimmable fat and bone increased ( ≤ 0.01) by 0.146 and 0.050 kg/d, respectively. These data illustrate improved saleable meat yields for calf-fed Holstein steers supplemented with ZH and provide the beef industry knowledge of fabrication yield changes throughout a wide range of harvest endpoints.

OBJECTIVE To investigate the effects of dietary supplementation with the β-adrenoceptor agonists ractopamine hydrochloride and zilpaterol hydrochloride on ECG and clinicopathologic variables of finishing beef steers. DESIGN Randomized controlled trial. ANIMALS 30 Angus steers. PROCEDURES Steers were grouped by body weight and randomly assigned to receive 1 of 3 diets for 23 days: a diet containing no additive (control diet) or a diet containing ractopamine hydrochloride (300 mg/steer/d) or zilpaterol hydrochloride (8.3 mg/kg [3.8 mg/lb] of feed on a dry-matter basis), beginning on day 0. Steers were instrumented with an ambulatory ECG monitor on days -2, 6, 13, and 23, and continuous recordings were obtained for 72, 24, 24, and 96 hours, respectively. At the time of instrumentation, blood samples were obtained for CBC and serum biochemical and blood lactate analysis. Electrocardiographic recordings were evaluated for mean heart rate and arrhythmia rates. RESULTS Steers fed zilpaterol or ractopamine had greater mean heart rates than those fed the control diet. Mean heart rates were within reference limits for all steers, with the exception of those in the ractopamine group on day 14, in which mean heart rate was high. No differences in arrhythmia rates were identified among the groups, nor were any differences identified when arrhythmias were classified as single, paired, or multiple (> 2) beats. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that dietary supplementation of cattle with ractopamine or zilpaterol at FDA-approved doses had no effect on arrhythmia rates but caused an increase in heart rate that remained within reference limits.

This study is a meta-analysis of the effects of the beta-agonists zilpaterol hydrochloride (ZH) and ractopamine hydrochloride (RAC) on feedlot performance, carcase characteristics of cattle and Warner Bratzler shear force (WBSF) of muscles. It was conducted to evaluate the effect of the use of these agents on beef production and meat quality and to provide data that would be useful in considerations on the effect of these agents on meat quality in Meat Standards Australia evaluations. We conducted a comprehensive literature search and study assessment using PubMed, Google Scholar, ScienceDirect, Scirus, and CAB and identification of other studies from reference lists in papers and searches. Searches were based on the key words: zilpaterol, zilmax, ractopamine, optaflexx, cattle and beef. Studies from theses obtained were included. Data were extracted from more than 50 comparisons for both agents and analysed using meta-analysis and meta-regression. Both agents markedly increased weight gain, hot carcase weight and longissimus muscle area and increased the efficiency of gain:feed. These effects were particularly large for ZH, however, fat thickness was decreased by ZH, but not RAC. Zilpaterol also markedly increased WBSF by 1.2 standard deviations and more than 0.8 kg, while RAC increased WBSF by 0.43 standard deviations and 0.2 kg. There is evidence in the ZH studies, in particular, of profound re-partitioning of nutrients from fat to protein depots. This work has provided critically needed information on the effects of ZH and RAC on production, efficiency and meat quality.

Ractopamine HCl and zilpaterol HCl, beta-agonists recently approved for use in feedlot cattle to improve performance traits and carcass leanness, were examined for their effects on fecal shedding of Escherichia coli O157:H7 in feedlot cattle. Fecal samples (n = 2,454) were obtained from four experiments (one ractopamine HCl, three zilpaterol HCl) over the course of a 3-year period, either by rectal palpation (ractopamine HCl experiment) or from pen-floor fecal pats. Samples were cultured quantitatively and qualitatively for E. coli O157:H7. No significant treatment differences were detected for fecal prevalence of E. coli O157:H7 in the ractopamine HCl experiment. Zilpaterol HCl feeding had no effect (P > 0.20) on fecal shedding in the first or second experiments, with overall E. coli O157:H7 prevalence relatively low (<7%). In the third zilpaterol HCl experiment, the percentage of fecal samples that were E. coli O157:H7 positive following qualitative culture was higher (P < 0.05) in the zilpaterol HCl treatment (10.3%) than for the control (6.1%). The current research showed minimal effects of beta-agonists on fecal shedding of E. coli O157:H7 and indicated that these compounds (fed immediately prior to slaughter) are not a cause for concern from a food safety standpoint.

Background: 4-[3,5-Bis (trimethylsilyl) benzamido] benzoic acid (TAC-101) is a novel retinobenzoic acid derivative which has a specific binding affinity to the retinoic acid receptors (RAR)alpha and RARbeta. Using time-dependent FACScan analysis, it was observed that TAC-101 induced apoptosis in a DLD-1 human colon cancer cell line. In this study, the induction of apoptosis-related proteins and the activities of caspases in a DLD-1 cell line under medication with TAC-101 were investigated., Materials and Methods: DLD-1 cells were cultured with different concentrations of TAC-101 for 12, 24 and 48 h. The expressions of Fas, TNF-R1, DR3, bcl-2, Bax and Bid were measured using a Western blot analysis. The activities of caspase-3, -8 and -9 were measured using a colorimetric protease assay kit., Results: The Western blot analysis showed that TAC-IO1 had almost no effect on the level of Bcl-2, Bax or Bid protein. Although TAC-101 did not change the expression of TNF-R1 and DR3, TAC-101 increased the expression of Fas in both a time- and a dose-dependent manner. A 3-fold increase in caspase-3 activity and a 1.5-fold increase in caspase-8 activity were observed in cells treated with TAC-101 in comparison to the control cells (p<0.01)., Conclusion: Our data indicate that the death receptor root of the apoptotic signal transduction in DLD-1 cells mainly participates in the apoptotic induction of TAC-101. Because the compounds inducing apoptotic activity are frequent targets of cancer therapy, TAC-101 may be a good candidate for use in the treatment of colon cancer.

Background: 4-[3,5-Bis (trimethylsilyl) benzamido] benzoic acid (TAC-101) is a novel retinobenzoic acid derivative, which has a specific binding affinity to the retinoic acid receptors (RAR)-alpha and -beta. Apoptotic induction by TAC-101 was investigated using a rat hepatic metastatic model of rat RCN-9 colon cancer cells in vivo and FACScan analysis with the DLD-1 human colon cancer cell line in vitro., Materials and Methods: Hepatic metastatic tumors were induced using intra-portal injection of RCN-9 cells into F344 rats in vivo. TAC-101 (8 mg/kg) was orally administered for 5 consecutive days a week for 4 weeks. Subsequently, hepatic tumors were counted after laparotomy. Apoptotic index (A.I.) in the hepatic tumors was evaluated using immunohistochemistry for single-stranded DNA. The proliferative index (P.I.), Fas and Fas ligand were also immunohistochemically evaluated. Moreover, evaluation of apoptosis by TAC-101 in vitro using FACScan analysis was performed in the DLD-1 human colon cancer cell line., Results: Oral administration of TAC-101 resulted in a significant inhibition of hepatic metastasis without weight loss of the rats. TAC-101 significantly decreased P. I. but increased A. I. in the hepatic metastatic tumors. TAC-101 did not affect the expression of Fas ligand, but obviously increased the expression of Fas in the metastatic tumors. Moreover, TAC-101 induced early apoptosis in DLD-1 cells in a time-dependent manner in vitro., Conclusion: These findings suggest that TAC-101 inhibits hepatic metastasis of colon cancer and induces apoptosis partially through enhanced Fas expression.

A-285222 (A-285) is a bis-trifluoromethyl-pyrazole (BTP), a novel class of immunosuppressive agents that inhibit NFAT activity in vitro in human and non-human primate cells through a calcineurin-independent mechanism. In this preliminary study, we treated cynomolgus monkeys with different doses of A-285 for several days. Blood was collected from all animals at different times during the study. From these samples, plasma concentrations of A-285 were measured by liquid chromatography/mass spectrometry (LC/MS), and intracellular T-cell production of the cytokines IL-2, IFN-gamma, and TNF-alpha was quantified by flow cytometry using a mitogen-stimulated whole blood assay. Marked inhibition of cytokine production occurred after administration of the first dose of A-285, and this effect was comparable to that of cyclosporine. While neurological toxic side effects were seen when the plasma concentration of A-285 exceeded 4 microg/ml, at lower plasma levels the drug was well tolerated over 2 weeks and its pharmacodynamic effects were sustained throughout this time.

A new series of azole derivatives of 1-triazolyl-4-trimethylsilyl-2-butanol and 1-triazolyl-5-trimethylsilyl-2-pentanol were synthesized and evaluated for fungicidal activities against rice blast, sheath blight, and powdery mildew on barley. The derivatives of 2,4-difluorobenzene exhibited high antifungal activities when applied by spray, but exhibited no fungicidal activity by submerged application.

TAC-101 (4-[3,5-bis(trimethylsilyl)benzamido]benzoic acid) is a novel, synthetic retinoid that is effective against liver metastases of human gastrointestinal cancer cells such as the human stomach carcinoma line AZ-521 in animal models, and is currently in use in phase I cancer trials. However, the mechanism of its antimetastatic action is still poorly understood. Tumor metastasis depends on angiogenesis, and various retinoids have been found to exhibit antiangiogenic activity. Based on these findings we here examined the antiangiogenic effects of TAC-101. Oral administration of TAC-101 (2-8 mg/kg/day) resulted in a drastic suppression of the AZ-521 cell-induced angiogenesis in a mouse dorsal air sac assay system, compared to the vehicle alone. Immunohistochemical analysis with antibody against the endothelial marker CD31 revealed a significant reduction in microvessel density in liver metastases from animals treated with TAC-101 (8 mg/kg p.o.), compared to liver metastases from the untreated control animals. The ability of TAC-101 (8 mg/kg p.o.) to prevent experimental liver metastasis of AZ-521 cells in athymic nude mice was comparable with that of the known angiogenesis inhibitor TNP-470 (30 mg/kg s.c.). TAC-101 also affected angiogenesis in chorioallantoic membranes and some functions of endothelial cells associated with angiogenesis, whereas the retinoid failed to suppress AZ-521 cell proliferation directly. These data suggest that the TAC-101 is an orally active antiangiogenic agent and that this antiangiogenic property may contribute to its efficacy against liver metastasis of human stomach cancer cells.

The anti-tumor and anti-metastatic effects of 4-[3,5-bis(trimethylsilyl)benzamido]benzoic acid (TAC-101) were investigated using our established lung cancer model. Orthotopic implantation of Lewis lung carcinoma (LLC) cells into the lung parenchyma produced a solitary tumor nodule in the lung followed by mediastinal lymph node metastasis. Daily oral administration of TAC-101 at doses ranging from 4 to 16 mg/kg resulted in a significant inhibition of lymphatic metastasis (inhibition rate=57 to 76%), while only the dose of 16 mg/kg significantly inhibited tumor growth at the implanted sites (inhibition rate=46%). Combined treatment with cis-diamminedichloroplatinum (CDDP) and TAC-101 (8 mg/kg, p.o., daily) enhanced the anti-tumor effect of CDDP (7 mg/kg, i.v., bolus) against both the growth of implanted tumor and lymphatic metastasis. In addition, this combined treatment significantly prolonged the survival time of LLC tumor-bearing mice as compared to treatment with each agent alone. The anti-activating protein-1 (AP-1) activity of TAC-101 caused inhibition of LLC cell invasion through the repression of expression of urokinase-type plasminogen activator and its receptor. The anti-invasive activity of TAC-101 may be involved in its in vivo anti-metastatic activity. These findings suggest that TAC-101 is a novel anti-cancer agent that may improve the therapeutic modalities for lung cancer patients with metastatic disease.

We demonstrated in this study that inhibition of intra-hepatic growth of colon cancer by TAC-101 is mediated by inhibition of angiogenesis. In vitro experiments showed that TAC-101 inhibited the proliferation of murine hepatic sinusoidal endothelial (HSE) cells induced by coculture with murine colon 26-L5 (L5) cells. HSE cell proliferation was also enhanced by conditioned medium of L5 cells (CM-L5), and this enhancement of proliferation was abrogated by anti-vascular endothelial growth factor antibody. CM-L5 also induced in vitro tube formation of HSE cells on Matri-gel, and this activity of CM-L5 was abrogated by TAC-101 in a concentration-dependent manner. On the other hand, p.o. administration of TAC-101 inhibited tumor-induced angiogenesis in vivo and decreased the weights of L5 tumors in the mouse liver. Reverse transcriptase-PCR analysis using in vivo tumor tissue suggested that repression of vascular endothelial growth factor expression by TAC-101 was associated with the antiangiogenic activity. TAC-101 alone and 5-fluorouracil (5-FU)/D,L-leucovorin (LV) significantly inhibited the intrahepatic growth of L5 tumors (P = 0.002 and 0.001, respectively), whereas 5-FU alone did not (P = 0.088). When TAC-101 was administered with 5-FU/LV, marked enhancement of antitumor activity was observed (95% inhibition; P<0.001). This enhanced antitumor effect was also observed in experiments using Co-3 human colon adenocarcinoma. Concurrent treatment with TAC-101 and 5-FU/LV and sequential treatment with 5-FU/LV followed by TAC-101 resulted in significant augmentation of antitumor activity against Co-3 (overall P = 0.007 and 0.015, respectively). These findings indicate that TAC-101 inhibits tumor angiogenesis and suggest that it may be effective against hepatic metastasis of colon cancer.

Retinoids, including all-trans-retinoic acid (ATRA), its isomers, and fifty synthetic retinoids (retinobenzoic acids), were tested for differentiation-inducing activity on human leukemia cell lines HL-60 and NB4. Binding activity of typical retinoids to nuclear retinoic acid receptors (RARs) was also investigated. A good linear correlation between the ED50 values of differentiation-inducing activity towards HL-60 cells and those towards NB4 cells was found. Binding activities of retinoids to RAR alpha and RAR beta also correlated well to the differentiation-inducing activities.

We have been examining a number of chemically modified mineral fibers, derived from amosite asbestos, by in vitro methods to clarify the role of the fiber surface in determining biological activity. The various fibers have identical size distributions but differ in their affinities for components of the cell membrane. They were treated with boiling toluene or chemically modified by treatment with alkyldimethylchlorosilanes (R = C8, C18) that react with free-surface hydroxyl groups to form the corresponding siloxanes. Fibers in MEM supplemented with 15% fetal calf serum were added to a suspension of V79-4 cells labeled with tritiated thymidine and the mixture was incubated. Aliquots of this mixture were spun down on a density gradient to determine the degree of cell-fiber interaction. At 37 degrees C native amosite (UICC standard) stuck to cells within 15 min of incubation, and the amount of sticking was maximum within 70 min. Decreasing the temperature decreased the amount of sticking, and at 20 degrees C no sticking was observable. The chemically modified amosite and the amosite treated with boiling toluene did not stick to the cells even after 70 min. Soaking the toluene-treated amosite with aqueous solutions at room temperature for 48 hr produced a material that had the same sticking properties as the original untreated fiber. These results indicate that the silanol content, and possibly the degree of hydration of the fiber surface, is important for a fiber to stick to a cell surface.

Suspensions of rat adrenocortical cells produce corticosterone as the major glucocorticoid. Cholesterol side-chain cleavage, the initial and rate-limiting step in the glucocorticoid biosynthetic pathway, is catalyzed by P-450scc. We have examined the effect of a variety of P-450scc inhibitors on corticosterone production by isolated rat adrenocortical cells. These inhibitors include reversible, noncovalently interacting inhibitors as well as mechanism-based inhibitors which irreversibly inactivate P-450scc in vitro. (20S)-22-nor-22-thiacholesterol and (22R)-22-aminocholesterol cause 50% inhibition of corticosterone production at 4 microM and 30 nM, respectively. Inhibition by these compounds was essentially not time-dependent. (20R)-20-(1-hexynyl)-pregn-5-en-3 beta, 20-diol and (20R)-20-(1,5-hexdiynyl)-pregn-5-en-3 beta, 20-diol at 10 microM inhibited corticosterone production in a time-dependent manner, resulting in 30% inhibition of corticosterone production during a 100-min incubation. (20S)-20-(2-trimethylsilyl ethyl)-pregn-5-en-3 beta, 20-diol inhibited in a strongly time-dependent manner. At 10 microM this compound irreversibly inhibited more than 90% of the side-chain cleavage capacity of the cell during a 40-min incubation. Cells treated with this steroid did not regain their capacity for side-chain cleavage after removal of free steroid. None of the inhibitors described above inhibited production of corticosterone by cells supplied with pregnenolone, the product of the P-450scc reaction. We suggest that the only significant effect of these compounds under these conditions is inhibition of the side-chain cleavage enzyme.

An antimicrobial-surface kinetic test which maximizes probability of cell-to-surface contact has been developed. The measurement of rate of kill by a nonleaching antimicrobial surface is based on the number of surviving bacterial cells at specific times of exposure to various amounts of total treated surface area of test substrate. This method gives information for direct comparison of rate of kill for a variety of antimicrobial surfaces in terms of rate of kill per square centimeter of surface area. Data obtained by this method can also give valuable dose response application information as an indication of the exponential efficiency of concentration in terms of treated surface area.