Oncogenicity testing of 2-ethylhexanol in Fischer 344 rats and B6C3F1 mice

An increasing number of studies, both experimental and epidemiologic, have provided evidence that filtering glaucoma surgery may be less effective than initially described. Of a number of risk factors for failure, duration and number of antiglaucoma drugs prior to surgery seem to play a critical role and highly accumulated antiglaucoma topical treatments significantly reduce success rates. Histopathological studies have confirmed that topically applied drugs may exert toxic effects to the corneoconjunctival surface, and induce chronic infraclinical inflammation, as shown by the presence of immune and inflammatory infiltrates in multitreated eyes. The origin of topical inflammation has not yet been clearly determined, but a common component of ophthalmic drugs, the benzalkonium chloride used as preservative in almost all antiglaucoma preparations, has shown strong evidence of toxicity. A number of questions remain to be investigated, but suppression of preservatives from chronically applied drugs should be a critical issue in the near future.     

Visual dysfunction in aged Fischer 344 rats

To investigate age-related changes in visual function in rats, male and female Fischer 344 (F344) rats at 30 months of age were examined electrophysiologically and histopathologically. The selection rate for the dark area in a T-shaped test box was 80% in aged rats, and the ability of light-dark discrimination was definitely depressed. Electroretinogram (ERG) was non-recordable in 25 out of the 28 eye balls examined, and amplitudes of the ERG a- and b-waves were markedly depressed in the remaining three eye balls. Histopathologic examination of the retina revealed marked atrophy of photoreceptor cells on the outer nuclear and photoreceptor layers; the change was less extensive in the retina of eye balls in which ERG was recordable. Immunohistochemically, increased reactivity to anti-glial fibrillary acid protein serum was observed in the retina of the aged animals. These results evidenced that the number of photoreceptor cells is decreased in age F344 rats, resulting in the reduced reactivity to light and the depressed light-dark discrimination.     

Distribution and excretion of radiolabelled tert-butylhydroquinone in Fischer 344 rats

Uniformly 14C-ring-labelled tert-butylhydroquinone (TBHQ) was diluted with non-radioactive TBHQ and administered orally (for excretion studies) to Fischer 344 rats. An average of 72.9% and 10.6% of the administered radioactivity was recovered in the urine and faeces, respectively, of male rats, and 77.3% and 8.2% in the urine and faeces, respectively, of female rats in 4 days. No significant sex-related differences were found in either excretion, tissue distribution or urinary metabolites of TBHQ-derived radiolabel. For distribution studies, intraperitoneal doses were administered to female rats, and tissue levels of radiolabel were determined at various times after dosing. The parent compound quickly disappeared from tissue in vivo. The highest concentrations of radiolabel were found in the liver and kidneys. The urinary metabolites consisted of conjugated TBHQ and unidentified polar substance(s).     

Muscle adaptations to hindlimb suspension in mature and old Fischer 344 rats

We examined skeletal and cardiac muscle responses of mature (8 mo) and old (23 mo) male Fischer 344 rats to 14 days of hindlimb suspension. Hexokinase (HK) and citrate synthase (CS) activities and GLUT-4 glucose transporter protein level, which are coregulated in many instances of altered neuromuscular activity, were analyzed in soleus (Sol), plantaris (PI), tibialis anterior (TA), extensor digitorum longus (EDL), and left ventricle. Protein content was significantly (P < 0.05) lower in all four hindlimb muscles after suspension compared with controls in both mature (21-44%) and old (17-43%) rats. Old rats exhibited significantly lower CS activities than mature rats for the Sol, Pl, and TA. HK activities were significantly lower in the old rats for the Pl (19%) and TA (33%), and GLUT-4 levels were lower in the old rats for the TA (38%) and EDL (24%) compared with the mature rats. Old age was also associated with a decrease in CS activity (12%) and an increase in HK activity (14%) in cardiac muscle. CS activities were lower in the Sol (20%) and EDL (18%) muscles from mature suspended rats and in the Sol (25%), Pl (27%), and EDL (25%) muscles from old suspended rats compared with corresponding controls. However, suspension was associated with significantly higher HK activities for all four hindlimb muscles examined, in both old (16-57%) and mature (10-43%) rats, and higher GLUT-4 concentrations in the TA muscles of the old rats (68%) but not the mature rats. These results indicate that old age is associated with decreased CS and HK activities and GLUT-4 protein concentration for several rat hindlimb muscles, and these variables are not coregulated during suspension. Finally, old rat skeletal muscle appears to respond to suspension to a similar or greater degree than mature rat muscle responds.     

Augmented mesenteric and renal vasoconstriction during exercise in senescent Fischer 344 rats

The purpose of this study was to test the hypothesis that vasoconstriction in the mesenteric and renal circulations is greater at both submaximal and maximal exercise intensities with advancing age. Arterial blood pressure, heart rate, and mesenteric, renal, and iliac (hindlimb) artery blood flow velocities were measured before and during graded treadmill exercise in mature (12 mo) and senescent (24 mo) male Fischer 344 rats. During treadmill running at mild, moderate, and maximal exercise intensities (approximately 45, 70, and 100% of maximal oxygen uptake), the increases in arterial pressure were similar in the mature and senescent animals, whereas heart rate rose less in the older group (P < 0.05). Mesenteric and renal flow velocities declined and vascular resistances increased from resting levels in both groups in response to graded exercise; however, the magnitudes of the increases in both mesenteric and renal vascular resistance were significantly augmented in the older rats at the moderate and maximal workloads. Hindlimb blood flow velocity increased and resistance declined from resting levels at each exercise intensity in both groups. In contrast to the visceral and renal adjustments, the magnitudes of the changes in both hindlimb flow and resistance were similar for the two age groups at all exercise intensities. These findings support the hypothesis that mesenteric and renal vasoconstriction is augmented in senescent Fischer 344 rats during exercise at moderate and maximal intensities but not at mild workloads. Despite these regional differences, the maintenance of arterial pressure is not altered at either submaximal or maximal exercise intensities with advancing age.     

Medicinal herb, Thonningia sanguinea protects against aflatoxin B1 acute hepatotoxicity in Fischer 344 rats

1. Thonningia sanguinea, a plant used prophylactically against bronchial asthma in Ghana was recently found to have antioxidative and hepatoprotective actions in our laboratory. 2. In this study, the effect of T. sanguinea extract on certain biochemical indices in serum and liver of Fischer 344 rats given a single intraperitoneal (i.p.) dose (1 mg/kg) of aflatoxin B1 (AFB1) was investigated. 3. Administration of AFB1 resulted in significant increases in serum alanine aminotransferase (ALT) and glutathione S-transferase (GST) levels and a significant decrease in aniline hydroxylase activity in liver microsomes. When T. sanguinea (5 ml/kg) was intraperitoneally administered to rats 12 h and 1 h before AFB1, liver injury was significantly reduced as seen in the decreased levels of serum ALT and serum GST. However, the decrease in aniline hydroxylase activity by AFB1 was not recovered but enhanced by T. sanguinea pre-treatment. 4. Kinetic analysis of cytochrome P450 activity of rat liver microsomes in vitro demonstrated that T. sanguinea inhibited aniline hydroxylase non-competitively suggesting depression of biotransformation of AFB1 to toxic metabolites. 5. The data indicate a hepatoprotective action of T. sanguinea against AFB1-induced liver injury.     

Intracellular calcium, DNase activity and myocyte apoptosis in aging Fischer 344 rats

Myocyte apoptosis increases with age in Fischer 344 rats, but the multiple molecular events implicated in this phenomenon remain to be identified. Several defects involving Ca2+ homeostasis, pH, and the expression of p53 and genes of the Bcl-2 protein family may contribute to the activation of myocyte death. Therefore, changes in intracellular pH, cytosolic Ca2+, DNase I and DNase II were measured in myocytes isolated by enzymatic digestion from rats of different ages. Moreover, the expression of p53, Bcl-2 and Bax in these cells was determined. Measurements of intracellular pH by BCECF fluorescence at 3, 12 and 24 months showed that this parameter did not change with age: 3 months, 7.20+/-0.05; 12 months, 7.21+/-0.07; 24 months, 7.18+/-0.09. In contrast, diastolic Ca2+ determined by the Fura 2-AM method increased progressively from 99.8+/-1.9 nm at 3 months to 136.3+/-9.6 nm at 24 months (P<0.001). Concurrently, DNase I activity evaluated by plasmid digestion assay in myocytes increased 3.2-fold from 3 to 24 months (P<0.02). Conversely, pH-dependent-DNase II remained essentially constant with age. Western blotting performed on ventricular myocytes did not detect significant changes in p53, Bax and Bcl-2 proteins with age. Similarly, immunocytochemically, the fraction of myocytes labeled by p53, Bax and Bcl-2 did not change from 3 to 24 months. In conclusion, myocyte aging is characterized by an increase in diastolic calcium which may activate DNase I triggering apoptosis, independently from the expression of p53, Bax and Bcl-2 in the cells.     

Development of the enhanced neural response to NaCl in Fischer 344 rats

Duplications of the esophagus or stomach alone are infrequent, and complete foregut duplication has only rarely been described. Most combined esophagogastric duplications present within the first year of life, and if communication with the normal alimentary tract does occur, it does so only either above or below the diaphragm. This report illustrates a case of continuous duplication of the esophagus and stomach with communication to the normal alimentary tract at both proximal and distal ends. Operative management and a review of the literature and embryology are described.     

Pathologic characterization of brown Norway, brown Norway x Fischer 344, and Fischer 344 x brown Norway rats with relation to age

The rat is a common laboratory animal utilized in a variety of investigations including experimental gerontology. Gerontologic investigations can be compromised when the differences observed when comparing young and old animals are actually differences between normal and disease states. It is of critical interest to know the pathology of the animals being studied and to understand the impact of these disease processes on the parameters being measured. The incidence and average age of occurrence for lesions have been characterized and are reported here for one inbred (Brown Norway) and two hybrid strains (Brown Norway x Fischer 344 and Fischer 344 x Brown Norway) of rat. Total lesion incidence functions as a biomaker of aging for all of the strains examined (p < or = .00001). These three genotypes have significantly lower incidence of several major pathologic processes (including glomerulonephritis, retinal atrophy, and leukemia) than do the Fischer 344 and the Wistar rats, two commonly utilized strains. Additionally, the BN and F344 x BN F1 hybrid attain 50% mortality at 130 and 146 weeks of age, respectively, which is significantly greater than the 103 weeks for the F344 rat. It is hoped that access to basic information on these three rat genotypes will increase their utilization by the community of gerontologic scientists.     

Time- and dose-dependent development of potassium bromate-induced tumors in male Fischer 344 rats

Potassium bromate (KBrO3) is a rodent carcinogen and a nephro- and neurotoxicant in humans. KBrO3 is used in cosmetics and food products and is a by-product of water disinfection by ozonization. KBrO3 is carcinogenic in the rat kidney, thyroid, and mesothelium and is a renal carcinogen in the male mouse. The present study was designed to investigate the relationship of time and dose to bromate-induced tumors in male Fischer 344 (F344) rats and to provide some insight into the development of these tumors. KBrO3 was dissolved in drinking water at nominal concentrations of 0, 0.02, 0.1, 0.2, and 0.4 g/L and administered to male F344 rats as the sole water source for 12, 26, 52, 78, or 100 wk. Renal cell tumors were present after 52 wk of treatment only in the high-dose group. Mesotheliomas developed after 52 wk of treatment on the tunica vaginalis. Mesotheliomas were present at sites other than the testicle after 78 wk of treatment, indicating that their origin was the testicular tunic. Thyroid follicular tumors were present as early as 26 wk in 1 rat each from the 0.1- and 0.2-g/L groups. The present study can be used as a basis for the determination of dose-time relationships of tumor development for a better understanding of KBrO3-induced cancer.     

Mesotheliomas of tunica vaginalis testis of Fischer 344 (F344) rats treated with acrylamide: a light and electron microscopy study

It is known that there are large temperature elevations in proximity to air bubbles during US (ultrasound) heating. The existence of tiny air bubbles in the target tissue may enhance the temperature elevation in US hyperthermia. To examine this hypothesis, phantom tissue experiments using an US contrast agent consisting of tiny air bubbles surrounded by a 5% (w/v) human albumin shell (Alb) were performed. As a phantom tissue, a 2 cm cube of beef was used. The phantom tissue was heated with or without the US contrast agent by an US hyperthermia device for 3 min. The heating device was operated at 1.5 MHz with the US intensity of 0.9 W/cm2. Physiological saline solution, iodized oil, and ethanol were used for control experiments. The effect of multiple needle punctures to the beef phantom was also examined. The temperature elevation rate (TER) was defined as the ratio of temperature elevation by heating with Alb or control materials to the temperature elevation by US heating alone. The TER of Alb was 1.7, whereas the TERs of the control materials and of the multiple needle punctures were approximately 1. The administration of Alb significantly increased the temperature in US hyperthermia. In addition, the heating efficiency of Alb was compared to the effect of an increase in the US intensity. Phantom tissue was heated at various US intensities. When the US intensity was increased from 0.9 to 1.8 W/cm2, the temperature elevated by approximately 1.7-fold. Thus, the effect of the administration of Alb was almost equivalent to the effect of increase in US power intensities from 0.9 to 1.8 W/cm2 in the present experimental settings. The results suggest that the US contrast agent can be a potential enhancer in US hyperthermia.     

Age and gender influence basal and stress-modulated hypothalamic-pituitary-thyroidal function in Fischer 344/N rats

To investigate possible gender- and age-associated changes of the hypothalamic-pituitary-thyroid (HPT) axis at baseline and during stress, we studied healthy young (3-month) and old (23-month) female 344/N Fischer rats at the basal state and after 2 h of immobilization (IMMO), in parallel to age-matched male rats. At baseline, there were no major differences on HPT axis functions between young female and male animals. Old age was associated with impaired central thyroid function in both genders, albeit to a much lesser extent in females than in males. Plasma prolactin (PRL) levels were similar in young females and males but were higher in old females than males. IMMO inhibited HPT axis functions in both genders in young, but not old animals. Thus, plasma TSH and hypothalamic TRH mRNA levels were decreased by IMMO in young, but not in old rats of both genders. IMMO increased plasma PRL in young and old males, but did not have any effect in young and old females. In summary, these data indicate that age and gender exert diverse effects on HPT axis functions at baseline and after stress.     

Age-related deficits in the cerebellar beta adrenergic signal transduction cascade in Fischer 344 rats

Localization of age-related deficits in the cerebellar beta adrenergic signal transduction cascade were investigated electrophysiologically using forskolin (FORSK) and adenosine-3',5'-cyclic monophosphothioate Sp-isomer (Sp-cAMPS) applied via pressure ejection from extracellular multibarreled glass electrodes to activate the transduction cascade. In young rats, 100 microM FORSK activated AC, and 100 microM Sp-cAMPS activated protein kinase A; thus, both increased GABAergic inhibition of Purkinje cell firing. In aged rats, however, 100 microM FORSK was unable to increase GABAergic inhibition of Purkinje cell firing. In addition, 1 mM 7 beta-decacetyl-7 beta-(gamma-N-methylpiperazino)butyryl-forskolin, an analog of FORSK, was also unable to increase GABAergic inhibition in aged rats. In contrast, Sp-cAMPS was able to increase GABAergic inhibition in aged rats, but higher doses were required than in young rats, Isoproterenol (ISO), a beta adrenergic agonist, was ineffective in increasing GABAergic inhibition of Purkinje firing in aged rats when tested alone, but ISO was effective in increasing Purkinje cell inhibition when ISO was tested with Sp-cAMPS. The results of this experiment indicate that one age-related deficit in the cerebellar beta adrenergic system occurs at the level of protein kinase A activation.     

Effect of voluntary exercise on maximal oxygen uptake in young female Fischer 344 rats

The effect of voluntary exercise on maximal oxygen uptake (VO2 max) was studied in young female Fischer 344 rats. After 10 weeks of wheel-running training, the absolute VO2 max and VO2 max relative to body mass increased without a decline in body mass. The running speed eliciting VO2 max, heart and soleus muscle mass, and succinate dehydrogenase (SDH) activity in the soleus muscle also increased. These results suggest that voluntary exercise is an effective means of increasing the aerobic exercise capacity of young female Fischer 344 rats.     

Studies of tamoxifen as a promoter of hepatocarcinogenesis in female Fischer F344 rats

Tamoxifen, an antiestrogen used in the treatment of breast cancer, was assessed for carcinogenic potential in the two-stage model of experimental hepatocarcinogenesis. Groups of female Fisher F344 rats were initiated with a non-necrogenic, subcarcinogenic dose of diethylnitrosamine (DEN; 10 mg/kg, po) and fed tamoxifen at a concentration of 250 mg per kg of AIN-76A diet for 6 or 15 months. The livers of these animals exhibited an increase in size and number of altered hepatic foci compared with those animals which were initiated with DEN but not exposed to tamoxifen. This finding indicates that tamoxifen may have a carcinogenic potential in the rat liver. After 6 months of treatment, neoplastic nodules were observed in 3/8 rats in the DEN-initiated, tamoxifen-treated group. In the initiated group provided with tamoxifen for 15 months, neoplastic nodules were observed in 7/8 rats and hepatocellular carcinomas in 3/8 rats. The serum level of tamoxifen in these rats was 200-300 ng/ml. The ratio of tamoxifen, 4-hydroxy tamoxifen, and N-desmethyl tamoxifen was 1:0.1:0.5-1 in the serum. When adjusted for age-related weight increases, the serum and liver levels of tamoxifen and its N-desmethyl metabolite did not change over the 15 months. In the rat liver, the level of tamoxifen and its N-desmethyl metabolite was 10-29 micrograms/g liver after 6 or 15 months of chronic dietary administration. The ratio of tamoxifen:4-hydroxy tamoxifen:N-desmethyl tamoxifen was 1:0.1.3-3.3 in the liver. Therefore, the liver had 20- to 30-fold more tamoxifen and 4-hydroxy tamoxifen and at least 100-fold more N-desmethyl tamoxifen than the serum (assuming 1 gram of tissue is equivalent to 1 ml of serum). These results indicate that tamoxifen is a promoting agent for the rat liver at serum levels found in patients given the usual therapeutic course of tamoxifen. The high concentrations of tamoxifen attained in the rat liver indicate that actions other than its known estrogenicity for liver could contribute to its promoting action. In addition, these results indicate that the pharmacodynamic differences in tamoxifen metabolism in rats and humans and at low versus high doses should be determined. Thus, the therapeutic indications for tamoxifen should be balanced by the potential risk it may present as a promoting agent in mammalian liver.     

Hippocampal interneurons expressing glutamic acid decarboxylase and calcium-binding proteins decrease with aging in Fischer 344 rats

Aging leads to alterations in the function and plasticity of hippocampal circuitry in addition to behavioral changes. To identify critical alterations in the substrate for inhibitory circuitry as a function of aging, we evaluated the numbers of hippocampal interneurons that were positive for glutamic acid decarboxylase and those that expressed calcium-binding proteins (parvalbumin, calbindin, and calretinin) in young adult (4-5 months old) and aged (23-25 months old) male Fischer 344 rats. Both the overall interneuron population and specific subpopulations of interneurons demonstrated a commensurate decline in numbers throughout the hippocampus with aging. Interneurons positive for glutamic acid decarboxylase were significantly depleted in the stratum radiatum of CA1, the strata oriens, radiatum and pyramidale of CA3, the dentate molecular layer, and the dentate hilus. Parvalbumin interneurons showed significant reductions in the strata oriens and pyramidale of CA1, the stratum pyramidale of CA3, and the dentate hilus. The reductions in calbindin interneurons were more pronounced than other calcium-binding protein-positive interneurons and were highly significant in the strata oriens and radiatum of both CA1 and CA3 subfields and in the dentate hilus. Calretinin interneurons were decreased significantly in the strata oriens and radiatum of CA3, in the dentate granule cell and molecular layers, and in the dentate hilus. However, the relative ratio of parvalbumin-, calbindin-, and calretinin-positive interneurons compared with glutamic acid decarboxylase-positive interneurons remained constant with aging, suggesting actual loss of interneurons expressing calcium-binding proteins with age. This loss contrasts with the reported preservation of pyramidal neurons with aging in the hippocampus. Functional decreases in inhibitory drive throughout the hippocampus may occur due to this loss, particularly alterations in the processing of feed-forward information through the hippocampus. In addition, such a profound alteration in interneuron number will likely alter inhibitory control of excitability and neuronal synchrony with behavioral states.     

Glucose transporter content and enzymes of metabolism in nerve-repair grafted muscle of aging Fischer 344 rats

Aging and grafting are associated with decreased ability of muscle to sustain power, likely reflecting diminished fuel availability. To assess mechanisms that may contribute to availability of glucose, we studied GLUT-1 and GLUT-4 protein as well as mRNA contents and enzymes of glucose metabolism in grafted and control medial gastrocnemius (MG) muscles of 6-, 12-, and 24-mo-old male Fischer 344 rats. There was no effect of age or grafting on MG GLUT-4 content. There was both an age- and graft-associated increase in GLUT-1 content (P = 0.0044 and 0.0063, respectively). There was no effect of aging or grafting on hexokinase and phosphofructokinase activity or on protein and glycogen content. Muscle mass and citrate synthase activity were significantly diminished with grafting. Citrate synthase activity was significantly greater in the 12-mo-old compared with the 6- and 24-mo-old animals. Grafting in combination with aging had no impact on any of the parameters measured. We conclude that diminished glucose transporter expression cannot explain the decreased ability of aged muscle to sustain power. In addition, we conclude that the diminished ability of the grafted MG muscle to sustain power may be explained, in part, by a decrease in energy available from oxidative metabolism.     

Sodium appetite after transection of the chorda tympani nerve in Wistar and Fischer 344 rats.

Acute sodium depletion in rats leads to dramatic increases in intake of hypertonic NaCl solutions, a behavior known as sodium appetite. The importance of signals conveyed by the chorda tympani (ChT) nerve to the expression of sodium appetite is unclear. The effects of bilateral ChT transection were examined on the short- and long-term response to sodium depletion in Wistar and Fischer 344 (F344) rat strains, because Wistar rats normally display a NaCl preference in the absence of need whereas F344 rats avoid NaCl. In both strains, sodium appetite after ChT transection and treatment with the diuretic furosemide was delayed and blunted or eliminated. The results suggest that signals conveyed by the ChT nerve are important in the expression of a sodium appetite. Effects on F344 rats are particularly interesting because ChT transection surgery appears to have opposite effects on NaCl intake depending on whether F344 rats are sodium replete or deplete. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

p53 mutations in hepatocellular carcinomas induced by a choline-devoid diet in male Fischer 344 rats

Analyses were performed on livers and hepatocellular carcinomas from male Fischer 344 rats fed a choline-devoid diet, to assess whether they carried alterations of the p53 tumor suppressor gene. The analyses consisted of immunoperoxidase staining of tissue sections with monoclonal antibodies to p53, Western blotting and cDNA sequencing. Immunostaining revealed the presence of mutant p53 proteins in 22/27 tumors analyzed and immunoblotting in 18/20. Immunochemical evidence was obtained that occurrence of the mutations precedes tumor development. cDNA sequencing was performed on 11 hepatocellular carcinomas that expressed mutant p53 gene proteins. Seven were found to contain point mutations within the 120-290 codon region of the gene, and one a microdeletion in the same region. No mutational hot spot was observed. It is concluded that mutations within the p53 gene, along with a c-myc gene amplification previously detected in these tumors, most likely contribute to the neoplastic transformation of liver cells in this nutritional model of hepatocarcinogenesis. The results are discussed also in view of recent literature on the presence of p53 mutations in human hepatocellular carcinomas.     

Role of TGF-beta in RA-induced cleft palate in CD-1 mice

Retinoic acid (RA) plays an important role in embryogenesis, by regulating morphogenesis, cell proliferation, differentiation, and extracellular matrix production. RA exposure on gestational day (GD) 12 in CD-1 mice results in delayed palatal shelf elevation and subsequent clefts in the secondary palate. Given the dynamic and complex nature of palate development, it is not surprising that this system is susceptible to changes in retinoid levels. There is evidence that experimental manipulation of retinoid status during development alters normal transforming growth factor-beta (TGF-beta) status. To study the role of perturbation in TGF-beta levels in RA-induced cleft palate, gravid CD-1 mice were treated with 70 mg/kg RA on GD 12. We examined changes in TGF-beta proteins and the steady-state level of TGF-beta mRNA within the first 24 hr after exposure. The interactions between RA and TGF-beta s were very complex. RA differentially regulated the mRNA and protein levels of TGF-beta 1. Changes in mRNA steady-state levels were rapid and transient in nature, indicating a direct mediation by RA. Differential regulation was evident, because RA treatment resulted in an increase in TGF-beta 1 mRNA steady levels followed by a decrease in the intracellular and extracellular forms of TGF-beta 1 protein. Moreover, the patterns of localization and levels of TGF-beta 2 and TGF-beta 3 proteins were not dramatically affected, although there was an increase in TGF-beta 3 mRNA steady-state levels. The increases in mRNA steady-state levels for TGF-beta 2 and TGF-beta 3, as for TGF-beta 1, were rapid and transient in nature, again arguing for direct mediation by RA. These data provide evidence for interactions between RA and TGF-beta s, and indicate that RA is capable of differentially regulating TGF-beta isoforms through processes involving different stages of TGF-beta synthesis and secretion. Further, changes in TGF-beta isoforms were observed prior to changes in mesenchyme morphology and must be considered as mediators of RA's effects on mesenchyme development.