Early indicators of response in biologically based risk assessment for nongenotoxic carcinogens.

The proposed existence of dose-response thresholds for nongenotoxic carcinogens has led to a major controversy in the risk extrapolation process. To resolve this debate, there has been a significant investment in mechanism-based risk assessment research. The ability to utilize this mechanistic research for risk assessment procedures is still limited and may not warrant the expense. Alternatively, an approach can be used to identify dose-response thresholds through the utilization of sensitive indicators of biological response. This approach does not rely upon a mechanistic framework for the development of pathology, is solely dependent on already existing technology, and takes into account the possibility of background levels of pathway activation. For this approach, sensitive biochemical responses need to be identified and linked to the introduction of the toxicant through dose response, by time of response, and, when possible, through a proposed biochemical mechanism. The weakness of this approach is that more sensitive unidentified responses may exist requiring that a safety factor of 10 be used to define a NOEL. For dioxin-like compounds, using a surrogate marker of response CYP1A1 induction, this approach yields an estimate of the acceptable daily intake of 5-50 fg/kg/day. This limit is remarkably similar to the results of the original EPA linear extrapolation (6 fg/kg/day). A similar approach can be used for other nongenotoxic carcinogens and the analysis can be completed within 1 year.

Role of a pineal cAMP-operated arylalkylamine N-acetyltransferase/14-3-3-binding switch in melatonin synthesis.

The daily rhythm in melatonin levels is controlled by cAMP through actions on the penultimate enzyme in melatonin synthesis, arylalkylamine N-acetyltransferase (AANAT; serotonin N-acetyltransferase, EC ). Results presented here describe a regulatory/binding sequence in AANAT that encodes a cAMP-operated binding switch through which cAMP-regulated protein kinase-catalyzed phosphorylation [RRHTLPAN --> RRHpTLPAN] promotes formation of a complex with 14-3-3 proteins. Formation of this AANAT/14-3-3 complex enhances melatonin production by shielding AANAT from dephosphorylation and/or proteolysis and by decreasing the K(m) for 5-hydroxytryptamine (serotonin). Similar switches could play a role in cAMP signal transduction in other biological systems.

Meniscal tissue regeneration using a collagenous biomaterial derived from porcine small intestine submucosa.

PURPOSE: The present investigation is a preliminary study designed to evaluate the use of a collagen-based biomaterial, chemically unaltered porcine small intestine submucosa (SIS), as a scaffold for meniscal tissue regeneration. TYPE OF STUDY: Basic research. METHODS: Surgical defects were created in the lateral menisci of 12 mature New Zealand white rabbits. The defects were repaired with a similarly shaped and sized wedge of a new collagenous biomaterial (SIS) and sutured in place. The opposite knees served as controls by creating a defect in the lateral meniscus without filling with SIS graft. Full cage activity was allowed until the animals were killed at 4, 12, and 24 weeks. RESULTS: At 4 weeks, the graft material retained its physical position and grossly appeared soft and translucent. Histologically, cellular elements had infiltrated between the laminates of the graft. At 12 weeks, the graft grossly appeared more solid and opaque. Histologically, the host meniscal fibrochondrocytes were seen streaming into the peripheral margin of the graft. Early repopulation of the graft with apparently differentiated meniscal tissue was observed. At 24 weeks, the meniscus defect was grossly healed across and looked virtually normal: the normal meniscal shape, contour, consistency, and color had been replicated. Histologically, the healing tissue showed infiltration of what appeared to be meniscal fibrochondrocytes and connective tissue resembling the host meniscal tissue. The graft was nearly totally replaced by host tissue. CONCLUSIONS: This pilot animal study demonstrates that the multilaminated collagenous graft is conducive for cellular repopulation with host meniscal elements, and, by 24 weeks, is capable of supporting complete healing of a large meniscal defect.

Chick pineal melatonin synthesis: light and cyclic AMP control abundance of serotonin N-acetyltransferase protein.

Melatonin production in the pineal gland is high at night and low during the day. This rhythm reflects circadian changes in the activity of serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87], the penultimate enzyme in melatonin synthesis. The rhythm is generated by an endogenous circadian clock. In the chick, a clock is located in the pinealocyte, which also contains two phototransduction systems. One controls melatonin production by adjusting the clock and the other acts distal to the clock, via cyclic AMP mechanisms, to switch melatonin synthesis on and off. Unlike the clock in these cells, cyclic AMP does not appear to regulate activity by altering AA-NAT mRNA levels. The major changes in AA-NAT mRNA levels induced by the clock seemed likely (but not certain) to generate comparable changes in AA-NAT protein levels and AA-NAT activity. Cyclic AMP might also regulate AA-NAT activity via changes in protein levels, or it might act via other mechanisms, including posttranslational changes affecting activity. We measured AA-NAT protein levels and enzyme activity in cultured chick pineal cells and found that they correlated well under all conditions. They rose and fell spontaneously with a circadian rhythm. They also rose in response to agents that increase cyclic AMP. They were raised by agents that increase cyclic AMP, such as forskolin, and lowered by agents that decrease cyclic AMP, such as light and norepinephrine. Thus, both the clock and cyclic AMP can control AA-NAT activity by altering the total amount of AA-NAT protein. Effects of proteosomal proteolysis inhibitors suggest that changes in AA-NAT protein levels, in turn, reflect changes in the rate at which the protein is destroyed by proteosomal proteolysis. It is likely that cyclic AMP-induced changes in AA-NAT protein levels mediate rapid changes in chick pineal AA-NAT activity. Our results indicate that light can rapidly regulate the abundance of a specific protein (AA-NAT) within a photoreceptive cell.

Natural melatonin 'knockdown' in C57BL/6J mice: rare mechanism truncates serotonin N-acetyltransferase.

Pineal melatonin synthesis (serotonin --> N-acetylserotonin --> melatonin) is severely compromised in most inbred strains of mice, in many cases because serotonin is not acetylated by serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT). We have found that in the C57BL/6J strain, AANAT mRNA encodes a severely truncated AANAT protein, because a pseudo-exon containing a stop codon is spliced in. This is the first identification of a natural mutation which knocks down melatonin synthesis. The decrease in melatonin signaling may have been a selective factor in the development of laboratory strains of mice because melatonin can inhibit reproduction and modify circadian rhythmicity.

Emergency removal of football equipment: a cadaveric cervical spine injury model.

STUDY OBJECTIVE: To determine the influence of football helmet and shoulder pads, alone or in combination, on alignment of the unstable cervical spine. METHODS: The alignment of the intact cervical spine in 8 cadavers was assessed radiographically under 4 different football equipment conditions: (1) no equipment, (2) helmet only, (3) helmet and shoulder pads, and (4) shoulder pads only. Each specimen was then surgically destabilized at C5-C6 to simulate a flexion-distraction injury. Repeat radiographs were obtained under the same 4 equipment conditions, and alignment of the unstable segment was analyzed. RESULTS: Before the destabilization, neutral alignment was maintained when both helmet and shoulder pads were in place. The "helmet only" condition caused a significant decrease in lordosis (mean, 9.6 +/- 4.7 degrees), whereas the "shoulder pads only" condition caused increased lordosis (13.6 +/- 6.3 degrees). After destabilization, the "helmet-only" condition demonstrated significant mean increases in C5-C6 forward angulation (16.5 +/- 8.6 degrees), posterior disc space height (3.8 +/- 2.3 mm), and dorsal element distraction (8.3 +/- 5.4 mm). CONCLUSION: Our flexion-distraction model demonstrated that immobilization of the neck-injured football player with only the helmet in place violates the principle of splinting the cervical spine in neutral alignment. By extrapolation to an extension-type injury, immobilization with only the shoulder pads left in place similarly violates this principle. In order to maintain a neutral position and minimize secondary injury to the cervical neural elements, the helmet and shoulder pads should be either both left on or both removed in the emergency setting.

Melatonin production: proteasomal proteolysis in serotonin N-acetyltransferase regulation.

The nocturnal increase in circulating melatonin in vertebrates is regulated by 10- to 100-fold increases in pineal serotonin N-acetyltransferase (AA-NAT) activity. Changes in the amount of AA-NAT protein were shown to parallel changes in AA-NAT activity. When neural stimulation was switched off by either light exposure or L-propranolol-induced beta-adrenergic blockade, both AA-NAT activity and protein decreased rapidly. Effects of L-propranolol were blocked in vitro by dibutyryl adenosine 3',5'-monophosphate (cAMP) or inhibitors of proteasomal proteolysis. This result indicates that adrenergic-cAMP regulation of AA-NAT is mediated by rapid reversible control of selective proteasomal proteolysis. Similar proteasome-based mechanisms may function widely as selective molecular switches in vertebrate neural systems.

The melatonin rhythm-generating enzyme: molecular regulation of serotonin N-acetyltransferase in the pineal gland.

A remarkably constant feature of vertebrate physiology is a daily rhythm of melatonin in the circulation, which serves as the hormonal signal of the daily light/dark cycle: melatonin levels are always elevated at night. The biochemical basis of this hormonal rhythm is one of the enzymes involved in melatonin synthesis in the pineal gland-the melatonin rhythm-generating enzyme-serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT, E.C. 2.3.1.87). In all vertebrates, enzyme activity is high at night. This reflects the influences of internal circadian clocks and of light. The dynamics of this enzyme are remarkable. The magnitude of the nocturnal increase in enzyme activity ranges from 7- to 150-fold on a species-to-species basis among vertebrates. In all cases the nocturnal levels of AA-NAT activity decrease very rapidly following exposure to light. A major advance in the study of the molecular basis of these changes was the cloning of cDNA encoding the enzyme. This has resulted in rapid progress in our understanding of the biology and structure of AA-NAT and how it is regulated. Several constant features of this enzyme have become apparent, including structural features, tissue distribution, and a close association of enzyme activity and protein. However, some remarkable differences among species in the molecular mechanisms involved in regulating the enzyme have been discovered. In sheep, AA-NAT mRNA levels show relatively little change over a 24-hour period and changes in AA-NAT activity are primarily regulated at the protein level. In the rat, AA-NAT is also regulated at a protein level; however, in addition, AA-NAT mRNA levels exhibit a 150-fold rhythm, which reflects cyclic AMP-dependent regulation of expression of the AA-NAT gene. In the chicken, cyclic AMP acts primarily at the protein level and a rhythm in AA-NAT mRNA is driven by a noncyclic AMP-dependent mechanism linked to the clock within the pineal gland. Finally, in the trout, AA-NAT mRNA levels show little change and activity is regulated by light acting directly on the pineal gland. The variety of mechanisms that have evolved among vertebrates to achieve the same goal-a rhythm in melatonin-underlines the important role melatonin plays as the hormonal signal of environmental lighting in vertebrates.

Isolation and characterization of a novel gene induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat liver.

The differential display technique was used to identify genes whose expression was regulated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Expression of a novel sequence was up-regulated in a dose-dependent fashion in liver of Sprague-Dawley male rats exposed to both chronic and acute treatment with TCDD, as measured by densitometry of Northern blot analyses (P < 0.01). A rapid amplification of cDNA ends (RACE) procedure was used to isolate a 1.8 kb cDNA from a rat liver cDNA preparation. This cloned cDNA, called 25-Dx, was sequenced and found to encode a peptide of 223 amino acids. In control rats, the 25-Dx gene was expressed at high levels in lung and liver. A hydrophobic domain of 14 residues followed by a proline-rich domain, both located in the N-terminal region, showed 71% homology with the transmembrane domain of the precursor for the interleukin-6 receptor and a conserved consensus sequence found in the cytokine/growth factor/prolactin receptor superfamily respectively.

Isolation of cDNAs representing dithiolethione-responsive genes.

Dithiolethiones inhibit tumorigenicity elicited by many structurally diverse carcinogens in numerous target tissues. These protective actions are associated with the induction of several carcinogen detoxification enzymes, some of which have only recently been discovered. In order to identify additional novel inducible detoxification response genes, a cDNA library was prepared from liver of rats treated with 1,2-dithiole-3-thione (D3T) and was screened by a differential hybridization method. Complementary DNA clones for several known D3T-inducible genes were isolated, such as epoxide hydrolase, aflatoxin B1-aldehyde reductase, quinone reductase and multiple subunits of glutathione S-transferase. Clones representing genes not previously associated with detoxification were isolated, including those for ferritin heavy and light subunits, ribosomal proteins L18a and S16 and two novel genes, termed dithiolethione-inducible genes (or DIG-1 and DIG-2). Levels of mRNA recognized by each clone were increased from 2- to 31-fold, with maximum induction between 6 and 30 h after treatment with D3T. Except for epoxide hydrolase, the kinetics of induction of each mRNA was coordinate with increased rates of gene transcription. However, based on the time of response to D3T, at least two sets of responsive genes were identified. One set of genes, including glutathione S-transferase Yp, aflatoxin B1-aldehyde reductase, quinone reductase and DIG-1, had low constitutive and highly inducible expression (approximately 20-fold) and the other, including glutathione S-transferase Ya and Yb, epoxide hydrolase, ferritin heavy and light subunits, ribosomal proteins L18a and S16 and DIG-2, had relatively high constitutive and modestly inducible expression (approximately 5-fold). The simplest explanation for this differential expression of D3T-inducible genes is that multiple regulatory mechanisms govern their response. The transcriptional activation of ferritin, ribosomal protein, DIG-1 and DIG-2 genes in conjunction with those of carcinogen detoxification enzymes suggests that they participate in the pleiotropic cellular defense response to dithiolethiones that inhibits chemically produced tumorigenesis.

A control system for cDNA enrichment reactions.

Most cDNA enrichment techniques involve technically complicated subtractive and hybridization reactions whose kinetics have not been characterized. We describe a control for cDNA enrichment procedures based on commercially available reagents, and we use this control to optimize a commonly used subtractive hybridization reaction. Using this control, we show that high-abundance transcripts can be efficiently removed following very short hybridization reactions. With the addition of unlabeled rat liver cDNA, we develop a system that mimics the reaction kinetics of complex cDNA pools. Because this system enables the measurement of specific vs. nonspecific hybridization and subtraction in the same tube and is an effective control for all steps in the cDNA enrichment procedure, it facilitates the development and optimization of novel cloning techniques for a desired abundance level of differentially expressed cDNAs.

Biologically bounded risk assessment for receptor-mediated nongenotoxic carcinogens.

We have developed a biologically bounded marginal effect model for use in risk assessment of human exposure to receptor-mediated nongenotoxic carcinogens. Schematically this model can be reduced to four components: CI, the absence of an observable biological response; CII, observable biochemical responses but no observable pathology; CIII, observable pathology; and CIV, both observable pathology and lethality. The inflection point in the marginal response curve between CI and CII is defined as the biologically evaluated scientifically tested no observable effect level (BESTNOEL). We demonstrate the utility of this approach by applying it to the well-studied nongenotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Using a well-developed mechanistic understanding of the initial interactions of TCDD with the cell, we justify the selection of the minimal effective dose for CYP1A1 mRNA induction as the BESTNOEL. With allowance for variation in human sensitivity to TCDD, the BESTNOEL is assigned a human liver tissue burden of approximately 0.25-25 ppt and an allowable daily intake level in the range of 15-1500 fg/kg/day. In the future, the BESTNOEL can help establish a lower boundary for acceptable extrapolation when using either statistical or biologically based attributable risk models.

Rat CYP1B1: an adrenal cytochrome P450 that exhibits sex-dependent expression in livers and kidneys of TCDD-treated animals.

The broad spectrum of biological responses associated with exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) is believed to be due to the alteration in expression of TCDD-inducible genes. The aim of this study was to investigate the effects of TCDD on the in vivo tissue-specific expression of the recently identified TCDD-inducible cytochrome P450 CYP1B1 [Sutter et al. (1994) J. Biol. Chem., 269, 13092-13099] in Sprague-Dawley rats. We cloned the 5.0 kb rat homolog of CYP1B1 from a TCDD-treated rat liver cDNA library and showed that the rat and human CYP1B1 predicted amino acid sequences are 80% identical. RNA hybridization analysis showed that CYP1B1 is constitutively expressed in the adrenal glands and also in the testes of untreated rats. This tissue distribution suggests that CYP1B1 may be a physiological steroid hydroxylase. Seventy-two hours post-administration of 25 micrograms/kg body wt TCDD by gavage, steady-state levels of the 5.1 kb CYP1B1 RNA were increased > 50-fold in liver, and to a lesser extent in kidneys, lung, heart and ovaries. Average CYP1B1 RNA levels were significantly higher in the kidneys and livers of TCDD-treated females than in those from similarly treated males. In contrast, no significant sex-difference was observed in the levels of CYP1A1 in these tissues in TCDD-treated animals. In Sprague-Dawley rats, TCDD is a more potent hepatocarcinogen in females than in males. The induction of CYP1B1 in TCDD rat liver may be a contributing factor to the carcinogenic action of this persistent environmental pollutant.

Prevention of heterotopic ossification with irradiation after total hip arthroplasty. Radiation therapy with a single dose of eight hundred centigray administered to a limited field.

Sixty-two hips in fifty-five patients who were considered to be at risk for postoperative heterotopic ossification were randomly divided into two groups: one received a single 800-centigray dose of limited-field radiation and the other, 1000 centigray of limited-field radiation in divided doses. The risk for heterotopic-bone formation was identified on the basis of previously described criteria, which included previous heterotopic ossification after an operation about the hip, hypertrophic osteoarthritis or post-traumatic osteoarthrosis characterized by formation of extensive osteophytes, radiographic evidence of diffuse idiopathic skeletal hyperostosis, ankylosing spondylitis, and male sex. The treatment portals excluded prosthetic surfaces that were intended for biological fixation by ingrowth of bone. At a minimum six-month follow-up, progression of heterotopic ossification had occurred in seven (21 per cent) of thirty-four hips in the first group and in six (21 per cent) of twenty-eight hips in the second group. The ossification had advanced more than one grade in only one hip. Extra-field ossification occurred in fifteen (43 per cent) of thirty-five hips that had not had previous heterotopic ossification. Since the time of the study, the treatment portal has been modified to include the lateral aspect of the greater trochanter, so that the risk of bursitis associated with ossification in this area is minimized. Single-dose limited-field radiation is effective for the prevention of heterotopic ossification, without compromise of early fixation of an uncemented implant.