Opioid-like adverse effects of tianeptine in male rats and mice.

RATIONALE: Tianeptine is a mu-opioid receptor (MOR) agonist with increasing reports of abuse in human populations. Preclinical data regarding the abuse potential and other opioid-like adverse effects of tianeptine at supratherapeutic doses are sparse. OBJECTIVES: The present study evaluated tianeptine in a rat model of abuse potential assessment and in mouse models of motor, gastrointestinal, and respiratory adverse effects. METHODS: Abuse potential was assessed in adult male Sprague-Dawley rats using an intracranial self-stimulation (ICSS) procedure to determine effects of acute and repeated tianeptine on responding for electrical brain stimulation. Male ICR mice were used to determine the effects of tianeptine in assays of locomotor behavior and gastrointestinal motility. Male Swiss-Webster mice were monitored for respiratory changes using whole-body plethysmography. RESULTS: In rats, acute tianeptine produced weak and delayed evidence for abuse-related ICSS facilitation at an intermediate dose (10 mg/kg, IP) and pronounced, naltrexone-preventable ICSS depression at a higher dose (32 mg/kg, IP). Repeated 7-day tianeptine (10 and 32 mg/kg/day, IP) produced no increase in abuse-related ICSS facilitation, only modest tolerance to ICSS depression, and no evidence of physical dependence. In mice, tianeptine produced dose-dependent, naltrexone-preventable locomotor activation. Tianeptine (100 mg/kg, SC) also significantly inhibited gastrointestinal motility and produced naloxone-reversible respiratory depression. CONCLUSIONS: Tianeptine presents as a MOR agonist with resistance to tolerance and dependence in our ICSS assay in rats, and it has lower abuse potential by this metric than many commonly abused opioids. Nonetheless, tianeptine produces MOR agonist-like acute adverse effects that include motor impairment, constipation, and respiratory depression.

Application of the paleolimnological method to assess metal contaminant distribution (As, Cu, Pb, Zn) in pulp mill stabilization basin sediments, Nova Scotia, Canada.

The paleolimnological method was used to decouple geogenic and anthropogenic metal (loids) contributions in a sediment stabilization basin (Boat Harbour) located in Nova Scotia, Canada. Boat Harbour has been impacted by industrial effluents discharged by a bleached kraft pulp mill (1967 to 2019) and a chlor-alkali plant (1971 to 1992). The former estuary now contains >577,000 m3 of unconsolidated sediment, impacted by inorganic and organic contaminants, including metal[loid]s, polycyclic aromatic hydrocarbons and polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans. Previous studies indicated significant knowledge gaps in our understanding of the spatial, stratigraphic, and temporal variation of sediment contamination. Twenty-five lakebed sediment gravity cores were obtained between 2016 and 2019 to determine spatiotemporal distribution of sediment As, Cu, Pb, and Zn concentrations which consistently exceeded guidelines for aquatic sediments. Results demonstrate there is no distinct spatial trend in metal concentrations despite point source effluent inputs. High and variable concentrations of Cu and Zn in contaminated sediment likely represent a combination of cation capture by highly organic sediment and influence of pulp mill on lakebed sediment chemistry. Elevated Pb in contaminated sediment is the result of atmospheric deposition from combustion of fossil fuels and bioaccumulation in effluent feedstock. Average sedimentation rate (1 cm every 3 years) is high compared to a nearby freshwater lake and is enhanced by increased nutrient loading and more productive water column conditions associated with effluent introduction. Temporal trends indicate significantly higher concentrations of Zn and Cu in top sediment samples consistent with changes in effluent treatment procedures as well as composition of effluent solids. Comparison of geochemistry of effluent influenced sediment and pre-effluent substate sediment at Boat Harbour to freshwater and marine reference was required to understand the degree to which geogenic and anthropogenic sources of metal(loids) have influenced effluent chemistry. This study demonstrates that undisturbed, time transgressive samples from both impacted sites and reference sites combined with non-destructive, rapid, small sample analytical techniques such as X-ray fluorescence, provide an accurate assessment of sediment metal contaminant distribution, data required to guide remediation and environmental effects monitoring and compliance.

The distribution and transport of lead over two centuries as recorded by lake sediments from northeastern North America.

We evaluated anthropogenic Pb deposition along a west-east transect from the Adirondack Mountains, New York, USA (ADIR) region, the Vermont-New Hampshire-Maine, USA (VT-NH-ME) region, and Nova Scotia, Canada (NS) region using 47 210Pb-dated lake sediment records. We used focus-corrected Pb inventories to evaluate cumulative deposition and breakpoint analysis to evaluate possible differences in timings among regions. Peak Pb concentrations decreased from west to east (ADIR region: 52-378 mg kg-1, VT-NH-ME region: 54-253 mg kg-1, NS: 38-140 mg kg-1). Cumulative deposition of anthropogenic Pb also decreased from west to east (ADIR region: 791-1344 mg m-2, VT-NH-ME region: 209-1206 mg m-2, NS: 52-421 mg m-2). The initiation of anthropogenic Pb deposition occurred progressively later along the same transect (ADIR region: 1869-1900, VT-NH-ME region: 1874-1905, NS region: 1901-1930). Previous lead isotope studies suggest that eastern Canadian Pb deposition over the past ~150 years has originated from a mix of both Canadian and U.S. sources. The results of this study indicate that anthropogenic Pb from sources west of the ADIR region were deposited in lesser amounts from west to east and/or Pb sources reflect less population density from west to east. The timing of the initiation of anthropogenic Pb deposition in the NS region suggests that Pb from gasoline may be an important source in this region.

Evaluating the utility of elemental measurements obtained from factory-calibrated field-portable X-Ray fluorescence units for aquatic sediments.

We assessed factory-calibrated field-portable X-ray fluorescence (pXRF) data quality for use with minimally-prepared aquatic sediments, including the precision of replicate pXRF measurements, accuracy of factory-calibrated pXRF values as compared to total digestion/ICP-OES concentrations, and comparability of calibrated pXRF values to extractable concentrations. Data quality levels for precision, accuracy, and comparability were not equivalent for element/analyzer combinations. All analyses of elements that were assessed for precision and accuracy on a single analyzer were both precise (<10% relative standard deviation) and accurate (r2 > 0.85) for K, Ca, Ti, Mn, Fe, and Zn. Calibrated pXRF values for Al, K, Ca, Ti, Mn, Fe, Cu, Zn, and Pb were within ∼10% relative difference of total digestion/ICP-OES concentrations. Calibrated pXRF values for Fe, Cu, Zn, As, and Pb were within ∼20% relative difference of extractable concentrations. Some elements had a higher level of data quality using specific analyzers, but in general, no pXRF analyzer had the highest level of data quality in all categories. Collectively, our data indicate that a wide range of factory-calibrated pXRF units are capable of providing high-quality total concentrations for the analysis of aquatic sediments.

Burn Hand or Finger Goniometric Measurements: Sum of the Isolated Parts and the Composite Whole.

Accurate assessment of hand function following a burn is important for patient impairment determination. Goniometric measurement of hand or finger range of motion (ROM) is typically done measuring individual finger joints with the adjacent joint in extension (isolated) or measuring the joints in a fist position (composite). The purpose of this study was to compare if the total flexion motion of the summed angles of the metacarpophalangeal, proximal interphalangeal, and distal interphalangeal joints in burned hands were equal when performed in an isolated vs a composite manner. Passive flexion ROM angles were collected prospectively and measured at the metacarpophalangeal, proximal interphalangeal, and distal interphalangeal with the adjacent joints extended to measure isolated angles and with the adjacent joints fully flexed for composite angles. Thumb joints were excluded. ROM for isolated and composite positions of eight fingers was compared individually and as an aggregate. Finger measurements from 145 adult patients were compared. The study population was predominately male (69%) with a mean age of 41 ± 16.6 years. Mean total burn size was 14.2 ± 13.2%. A total of 739 fingers contributed 2217 joint ROM comparisons. Aggregate analysis of isolated ROM was 235.5° ± 52.1° compared with composite ROM of 226.8° ± 53.2° (P < .0001). Individual fingers showed significant differences between the two measurement methods as well (P ≤ .0040). The methods used to measure hand or finger ROM profoundly influence how hand impairment is reported. Measurement of isolated joint angles results in greater ROM values compared with composite angles, which are often more relevant for functional hand positions. Therefore, composite angles are recommended.

Profile of Patients Without Burn Scar Contracture Development.

Burn scar contractures (BSCs) are a frequently recognized problem for survivors of burn injury. In the burn literature, many reports focus on the frequency and factors associated with the BSC development. To the contrary, few burn rehabilitation publications report on patients who are able to successfully avoid developing BSC. From a prospective, multicenter study, data were extracted and reviewed on a group of 56 adult burn survivors who were discharged from their acute hospitalization without any measured BSCs. Forty-three variables with a recognized or presumed association with the development of BSCs were analyzed and are reported. Highlighted features of the noncontracted group included being an adult male with an educated background and few associated physical, medical, or social problems. The group had relatively small burn sizes that nonetheless required hospitalization. Despite the overall TBSA, the majority of the burn areas required skin grafting, although this area also represented a small area. The patient group had a longer than expected hospital stay. Rehabilitation was provided to patients on 80% of their hospital days. In addition, patients received sufficient rehabilitation treatment based on the number of cutaneous functional units involved in the burn injury. Patients were judged to have a high pain tolerance and compliant with rehabilitation. The results of this study document the clinical circumstances that patients with burn injury can be discharged from their acute hospitalization with the development of BSC. This study challenges the rehabilitation personnel to expand the upper limit of burn severity that can result in similar positive outcomes.

Sensitization of enteric neurons to morphine by HIV-1 Tat protein.

BACKGROUND: Gastrointestinal (GI) dysfunction is a major cause of morbidity in acquired immunodeficiency syndrome (AIDS). HIV-1-induced neuropathogenesis is significantly enhanced by opiate abuse, which increases proinflammatory chemokine/cytokine release, the production of reactive species, glial reactivity, and neuronal injury in the central nervous system. Despite marked interactions in the gut, little is known about the effects of HIV-1 in combination with opiate use on the enteric nervous system. METHODS: To explore HIV-opiate interactions in myenteric neurons, the effects of Tat ± morphine (0.03, 0.3, and 3 µM) were examined in isolated neurons from doxycycline- (DOX-) inducible HIV-1 Tat(1-86) transgenic mice or following in vitro Tat 100 nM exposure (>6 h). KEY RESULTS: Current clamp recordings demonstrated increased neuronal excitability in neurons of inducible Tat(+) mice (Tat+/DOX) compared to control Tat-/DOX mice. In neurons from Tat+/DOX, but not from Tat-/DOX mice, 0.03 µM morphine significantly reduced neuronal excitability, fast transient and late long-lasting sodium currents. There was a significant leftward shift in V(0.5) of inactivation following exposure to 0.03 µM morphine, with a 50% decrease in availability of sodium channels at -100 mV. Similar effects were noted with in vitro Tat exposure in the presence of 0.3 µM morphine. Additionally, GI motility was significantly more sensitive to morphine in Tat(+) mice than Tat(-) mice. CONCLUSIONS & INFERENCES: Overall, these data suggest that the sensitivity of enteric neurons to morphine is enhanced in the presence of Tat. Opiates and HIV-1 may uniquely interact to exacerbate the deleterious effects of HIV-1-infection and opiate exposure on GI function.

Site and mechanism of morphine tolerance in the gastrointestinal tract.

Opioid-induced constipation is a major clinical problem. The effects of morphine, and other narcotics, on the gastrointestinal tract persist over long-term use thus limiting the clinical benefit of these excellent pain relievers. The effects of opioids in the gut, including morphine, are largely mediated by the µ-opioid receptors at the soma and nerve terminals of enteric neurons. Recent studies demonstrate that regional differences exist in both acute and chronic morphine along the gastrointestinal tract. While tolerance develops to the analgesic effects and upper gastrointestinal motility upon repeated morphine administration, tolerance does not develop in the colon with chronic opioids resulting in persistent constipation. Here, we review the mechanisms by which tolerance develops in the small but not the large intestine. The regional differences lie in the signaling and regulation of the µ-opioid receptor in the various segments of the gastrointestinal tract. The differential role of ß-arrestin2 in tolerance development between central and enteric neurons defines the potential for therapeutic approaches in developing ligands with analgesic properties and minimal constipating effects.

Reversal of morphine analgesic tolerance by ethanol in the mouse.

The chronic use of opioids in humans, accompanied by the development of tolerance, is a dangerous phenomenon in its own right. However, chronic opioid use is often made more dangerous by the coconsumption of other substances. It has been observed that the blood level of opioids in postmortem analyses of addicts, who consumed ethanol along with the opioid, was much less than that observed in individuals who died from opioids alone. This relationship between ethanol and opioids led us to investigate the hypothesis that ethanol alters tolerance to opioids. In the present study, we report that ethanol significantly and dose-dependently reduced the antinociceptive tolerance produced by morphine and the cross-tolerance between [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) and morphine in the mouse tail-flick test. The reversal of morphine tolerance was partially blocked by both the gamma receptor blocker bicuculline and by the γ-aminobutyric acid (GABA)(B) receptor blocker phaclofen and the administration of both inhibitors completely reversed the effects of ethanol on morphine tolerance. Diazepam, like ethanol, decreased morphine tolerance. However, this inhibition was reversed by the GABA(A) antagonist bicuculline but not by the GABA(B) antagonist phaclofen. These findings have important implications for individuals who abuse opioids and ethanol as well as suggest a mechanism to reduce the amount of opioid needed in chronic pain treatment.

The effect of protein kinase C and G protein-coupled receptor kinase inhibition on tolerance induced by mu-opioid agonists of different efficacy.

Differences in the mechanisms underlying tolerance and mu-opioid receptor desensitization resulting from exposure to opioid agonists of different efficacy have been suggested previously. The objective of this study was to determine the effects of protein kinase C (PKC) and G protein-coupled receptor kinase (GRK) inhibition on antinociceptive tolerance in vivo to opioid agonists of different efficacy. A rapid (8-h) tolerance-induction model was used where each opioid was repeatedly administered to naive mice. Animals were then challenged with the opioid after injection of a kinase inhibitor to determine its effects on the level of tolerance. Tolerance to meperidine, morphine, or fentanyl was fully reversed by the PKC inhibitor 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)carbazole (Gö6976). However, in vivo tolerance to [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) was not reversed by PKC inhibition. The novel small-molecule GRK inhibitors beta-adrenergic receptor kinase 1 inhibitor and 2-(8-[(dimethylamino) methyl]-6,7,8,9-tetrahydropyridol[1,2-a]indol-3-yl)-3-(1-methylindol-3-yl)maleimide (Ro 32-0432) did not reverse the tolerance to meperidine, fentanyl, or morphine but did reverse the tolerance to DAMGO. To correlate GRK-dependent DAMGO-induced tolerance with mu-opioid receptor desensitization, we used in vitro whole-cell patch-clamp recording from mouse locus coeruleus neurons and observed that the GRK inhibitors reduced DAMGO-induced desensitization of mu-opioid receptors, whereas the PKC inhibitor had no effect. These results suggest that tolerance induced by low- and moderate-efficacy mu-opioid receptor agonists is dependent on PKC, whereas tolerance induced by the high-efficacy agonist DAMGO is dependent on GRK.

Involvement of PKC alpha and G-protein-coupled receptor kinase 2 in agonist-selective desensitization of mu-opioid receptors in mature brain neurons.

BACKGROUND AND PURPOSE: The ability of an agonist to induce desensitization of the mu-opioid receptor (MOR) depends upon the agonist used. Furthermore, previous data suggest that the intracellular mechanisms underlying desensitization may be agonist-specific. We investigated the mechanisms underlying MOR desensitization, in adult mammalian neurons, caused by morphine (a partial agonist in this system) and DAMGO (a high-efficacy agonist). EXPERIMENTAL APPROACH: MOR function was measured in locus coeruleus neurons, by using whole-cell patch-clamp electrophysiology, in rat and mouse brain slices (both wild-type and protein kinase C (PKC)alpha knockout mice). Specific isoforms of PKC were inhibited by using inhibitors of the receptors for activated C-kinase (RACK), and in vivo viral-mediated gene-transfer was used to transfect neurons with dominant negative mutants (DNMs) of specific G-protein-coupled receptor kinases (GRKs). KEY RESULTS: Morphine-induced desensitization was attenuated by using RACK inhibitors that inhibit PKCalpha, but not by other isoform-specific inhibitors. Further, the PKC component of morphine-induced desensitization was absent in locus coeruleus neurons from PKCalpha knockout mice. The PKC-enhanced morphine-induced desensitization was not affected by over-expression of a GRK2 dominant negative mutant (GRK2 DNM). In contrast, DAMGO-induced MOR desensitization was independent of PKC activity but was reduced by over-expression of the GRK2 DNM but not by that of a GRK6 DNM. CONCLUSIONS AND IMPLICATIONS: In mature mammalian neurons, different MOR agonists can induce MOR desensitization by different mechanisms, morphine by a PKCalpha-mediated, heterologous mechanism and DAMGO by a GRK-mediated, homologous mechanism. These data represent functional selectivity at the level of receptor desensitization.

Role of protein kinase C and mu-opioid receptor (MOPr) desensitization in tolerance to morphine in rat locus coeruleus neurons.

In morphine tolerance a key question that remains to be answered is whether mu-opioid receptor (MOPr) desensitization contributes to morphine tolerance, and if so by what cellular mechanisms. Here we demonstrate that MOPr desensitization can be observed in single rat brainstem locus coeruleus (LC) neurons following either prolonged (> 4 h) exposure to morphine in vitro or following treatment of animals with morphine in vivo for 3 days. Analysis of receptor function by an operational model indicated that with either treatment morphine could induce a profound degree (70-80%) of loss of receptor function. Ongoing PKC activity in the MOPr-expressing neurons themselves, primarily by PKCalpha, was required to maintain morphine-induced MOPr desensitization, because exposure to PKC inhibitors for only the last 30-50 min of exposure to morphine reduced the MOPr desensitization that was induced both in vitro and in vivo. The presence of morphine was also required for maintenance of desensitization, as washout of morphine for > 2 h reversed MOPr desensitization. MOPr desensitization was homologous, as there was no change in alpha(2)-adrenoceptor or ORL1 receptor function. These results demonstrate that prolonged morphine treatment induces extensive homologous desensitization of MOPrs in mature neurons, that this desensitization has a significant PKC-dependent component and that this desensitization underlies the maintenance of morphine tolerance.

Arrhenius relationships from the molecule and cell to the clinic.

There are great differences in heat sensitivity between different cell types and tissues. However, for an isoeffect induced in a specific cell type or tissue by heating for different durations at different temperatures varying from 43-44 degrees C up to about 57 degrees C, the duration of heating must be increased by a factor of about 2 (R value) when the temperature is decreased by 1 degrees C. This same time-temperature relationship has been observed for heat inactivation of proteins, and changing only one amino acid out of 253 can shift the temperature for a given amount of protein denaturation from 46 degrees C to either 43 or 49 degrees C. For cytotoxic temperatures <43-44 degrees C, R for mammalian cells and tissues is about 4-6. Many factors change the absolute heat sensitivity of mammalian cells by about 1 degrees C, but these factors have little effect on Rs, although the transition in R at 43-44 degrees C may be eliminated or shifted by about 1 degrees C. R for heat radiosensitization are similar to those above for heat cytotoxicity, but Rs for heat chemosensitization are much smaller (usually about 1.1-1.2). In practically all of the clinical trials that have been conducted, heat and radiation have been separated by 30-60 min, for which the primary effect should be heat cytotoxicity and not heat radiosensitization. Data are presented showing the clinical application of the thermal isoeffect dose (TID) concept in which different heating protocols for different times at different temperatures are converted into equivalent minutes (equiv) min at 43 degrees C (EM(43)). For several heat treatments in the clinic, the TIDs for each treatment can be added to give a cumulative equiv min at 43 degrees C, namely, CEM(43). This TID concept was applied by Oleson et al. in a retrospective analysis of clinical data, with the intent of using this approach prospectively to guide future clinical studies. Considerations of laboratory data and the large variations in temperature distributions observed in human tumors indicate that thermal tolerance, which has been observed for mammalian cells for both heat killing and heat radiosensitization, probably is not very important in the clinic. However, if thermal tolerance did occur in the clinical trials in which fractionation schemes were varied, it probably would not have been detected because with only the two-three-fold change in treatment time that occurs when comparing one versus two fractions per week, or three versus six total fractions, little difference would be expected in the response of the tumors since both thermal doses were extremely low on the dose-response curve. Data are shown which indicate that in order to test for thermal tolerance in the clinic and to have a successful phase III trial, the thermal dose should be increased about five-fold compared with what has been achieved in previous clinical trials. This increase in thermal dose could be achieved by increasing the temperature about 1.5 degrees C (from 39.5 to 41.0 degrees C in 90% of the tumor) or by increasing the total treatment time about five-fold. The estimate is that 90% of the tumor should receive a cumulative thermal dose (CEM(43)) of at least 25; this is abbreviated as a CEM(43) T(90) of 25. This value of 25 compares with 5 observed by Oleson et al. in their soft tissue sarcoma study. Arguments also are presented that thermal doses much higher than the CEM(43) T(90) induce the hyperthermic damage that causes the tumors to respond, and that the minimum CEM(43) T(90) of 25 only predicts which tumors that receive a certain minimal thermal dose in <90% of the regions of the tumors will respond. For example, in addition to a minimal CEM(43) T(90) of 25 a minimum CEM(43) T(50) of about 400 also may be required for a response. Finally, continuous heating for approximately 2 days at about 41 degrees C during either interstitial low dose-rate irradiation or fractionated high dose-rate irradiation, which we estimate could give a CEM(43) of 75, should be considered in order to enhance heat radiosensitization of the tumor as well as heat cytotoxicity. In order to exploit the use of hyperthermia in the clinic, we need a better understanding of the biology and physiology of heat effects in tumors and various normal tissues. As an example of an approach for mechanistic studies, one specific study is described which demonstrates that damage to the centrosome of CHO cells heated during G(1) causes irregular divisions that result in multinucleated cells that do not continue dividing to form colonies. This may or may not be relevant for heat damage in vivo. However, since normal tissues vary in thermal sensitivity by a factor of 10, similar approaches are needed to describe the fundamental lethal events that occur in the cells comprising the different tissues.

Opposition splint for partial thumb amputation: a case study measuring disability before and after splint use.

STUDY DESIGN: Case report. INTRODUCTION: A combined burn and a partial amputation can be extremely debilitating as the thumb constitutes 40% of the entire hand when evaluating functional impairment. PURPOSE OF THE STUDY: Measure disability with and without opposition splint use after partial thumb amputation due to a burn. METHODS: Impairment and disability measures were completed at discharge from the hospital and subsequently during outpatient follow-up visits while wearing and not wearing a thumb opposition splint at 3, 6, 8, and 15 months. Comparisons between disability and impairment scores were assessed over time. RESULTS: The difference between DASH scores with and without using the splint were 25 at 3 months, 16 at 6 months, 10 at 8 months, and 12 at 15 months. CONCLUSIONS: Splint use in this case demonstrated clinically significant changes over time with minimal changes in impairment indicating enhanced function and improved patient perception of disability. LEVEL OF EVIDENCE: 4.

Region-dependent attenuation of mu opioid receptor-mediated G-protein activation in mouse CNS as a function of morphine tolerance.

BACKGROUND AND PURPOSE: Chronic morphine administration produces tolerance in vivo and attenuation of mu opioid receptor (MOR)-mediated G-protein activation measured in vitro, but the relationship between these adaptations is not clear. The present study examined MOR-mediated G-protein activation in the CNS of mice with different levels of morphine tolerance. EXPERIMENTAL APPROACH: Mice were implanted with morphine pellets, with or without supplemental morphine injections, to induce differing levels of tolerance as determined by a range of MOR-mediated behaviours. MOR function was measured using agonist-stimulated [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) and receptor binding throughout the CNS. KEY RESULTS: Morphine pellet implantation produced 6-12-fold tolerance in antinociceptive assays, hypothermia and Straub tail, as measured by the ratio of morphine ED(50) values between morphine-treated and control groups. Pellet implantation plus supplemental injections produced 25-50-fold tolerance in these tests. In morphine pellet-implanted mice, MOR-stimulated [(35)S]GTPgammaS binding was significantly reduced only in the nucleus tractus solitarius (NTS) and spinal cord dorsal horn in tissue sections from morphine pellet-implanted mice. In contrast, MOR-stimulated [(35)S]GTPgammaS binding was significantly decreased in most regions examined in morphine pellet+morphine injected mice, including nucleus accumbens, caudate-putamen, periaqueductal gray, parabrachial nucleus, NTS and spinal cord. CONCLUSIONS AND IMPLICATIONS: Tolerance and the regional pattern of apparent MOR desensitization were influenced positively by the level of morphine exposure. These results indicate that desensitization of MOR-mediated G-protein activity is more regionally widespread upon induction of high levels of tolerance, suggesting that this response contributes more to high than low levels of tolerance to CNS-mediated effects of morphine.

Carcinogenic effects of hyperthermia.

The purpose of this paper is to assess the evidence for and against the premise that hyperthermia is carcinogenic. The paper is one of several published in this issue of the International Journal of Hyperthermia on the subject of the health risks of hyperthermia. The motivation for this issue of the journal was the result of a World Health Organization workshop that dealt with this issue, as it relates to exposure of the population to RF fields. Since hyperthermia can be a natural consequence of such exposures, the health risks of hyperthermia are relevant in this context. Particularly in the case of carcinogenesis, it is necessary to provide a brief overview of the data that have been generated to examine the carcinogenic risks of RF exposure, so that these results can be compared with studies that have examined the carcinogenic risks of hyperthermia. For this reason, the paper is organized into three sections dealing with: (1) effects of heat on DNA damage/repair and mutations, (2) in vivo studies evaluating the carcinogenic potential of heat alone and combined with other carcinogens, and (3) in vivo studies involving RF exposures. The bulk of the data presented indicate that hyperthermia alone is not carcinogenic. If hyperthermia occurs in the presence of exposure to known carcinogens, such as radiation or chemical carcinogens there is the potential for modulation of carcinogenic effects of those agents. In some circumstances, hyperthermia can actually protect against tumour formation. In other instances, hyperthermia clearly increases incidence of tumour formation, but this occurs following thermal exposures (several degrees C temperature rise for up to 1 h or more) and radiation (therapeutic levels as for treatment of cancer) or chemical carcinogen doses higher than would be encountered by the general population. The extrapolation of these results to the general population, where radiation exposure levels would be at background and temperature rise from incidental RF exposure, such as cell phones (which are estimated to cause no more than 0.1 degrees C temperature rise) is not recommended. Current evidence indicates that the temperature elevations resulting from RF exposure are not carcinogenic. Caution should be used in situations where exposure to known carcinogens is combined with thermal exposures high enough to cause tissue damage. A summary of thermal thresholds for tissue damage from hyperthermia is presented in another paper in this special issue (Dewhirst et al.). No data exist that examine the carcinogenic risks of chronic thermal exposures below the threshold for detectable tissue damage, either alone or in combination with known carcinogens. This is an important goal for future research.

Hyperthermic teratogenicity, thermal dose and diagnostic ultrasound during pregnancy: implications of new standards on tissue heating.

Hyperthermia is a recognized teratogen in mammalian laboratory animals and is a suspected teratogen for humans. The purpose of this synopsis is to reanalyse existing data on hyperthermia-induced teratogenic effects in experimental mammalian systems in terms of a thermal dose (temperature:time) concept, and then to illustrate the utility of this concept to human situations involving potential thermal increments to post-implantation embryos and foetuses. For example, the threshold temperature elevation for hyperthermia-induced teratogenic effects in experimental mammals is estimated (but not rigorously tested) to be approximately 1.5 degrees C above core values for exposures of long duration, possibly with a thermal dose of approximately 5 min duration or more at 4 degrees C. This level of tissue temperature increment is within the capability of some modern diagnostic ultrasound (DUS) devices sold within the USA and abroad. Epidemiological studies have not indicated any hazard from the use of DUS, but such studies are limited in sensitivity and were conducted with DUS devices whose acoustic outputs were relatively low compared to those presently available. After a regulatory change that allowed for substantially increased acoustic outputs, modern DUS devices were mandated to provide the user with on-screen information (the Thermal Index, or 'TI') about ultrasound-induced temperature increments in the target tissue. The TI is generally accurate to within a factor of 2, but the factor may be as high as 6 in certain obstetric settings. Thus, informed use of and attention to the TI is strongly advised, with this admonition gaining increased emphasis if the present regulations regarding allowable acoustic outputs of DUS devices were to be further relaxed or eliminated.

Mutations induced in the HPRT gene by X-irradiation during G(1) or S: analysis of base pair alterations, small deletions, and splice errors.

Reverse transcriptase PCR was performed with mRNA obtained from HPRT mutants that had base pair alterations, or small deletions or insertions <20bp. The frequencies of mutants yielding RT-PCR products (mRNA) were the same when human EJ30 cells were irradiated in G(1) or S (3-4-fold higher for 6 than 3Gy). However, the frequencies of mutants that did not yield RT-PCR products were approximately 10-fold higher in the cells irradiated in G(1) than in those irradiated in S. Sequence analysis of RT-PCR products and genomic DNA showed that 40% of the RT-PCR products had splice errors (one or more exons not spliced into mRNA), with 64% of them due to 1-17bp deletions. Also, the distributions of molecular alterations in exons, acceptor sites, and donor sites for mutants having splice errors (observed in this study and reported by others) were similar to those reported for mutants not yielding RT-PCR products (isolated from Russian cosmonauts). In addition, we have found previously that large deletions which eliminated 1-9 exons were preferentially induced in G(1). Therefore, we postulate that the preferential induction of mutants not yielding mRNA is due primarily to splice errors that result from deletions preferentially induced during G(1). These splice errors would then result either in no message or a message that is rapidly degraded.

Computerized video time-lapse analysis of apoptosis of REC:Myc cells X-irradiated in different phases of the cell cycle.

Asynchronous rat embryo cells expressing Myc were followed in 50 fields by computerized video time lapse (CVTL) for three to four cycles before irradiation (4 Gy) and then for 6-7 days thereafter. Pedigrees were constructed for single cells that had been irradiated in different parts of the cycle, i.e. at different times after they were born. Over 95% of the cell death occurred by postmitotic apoptosis after the cells and their progeny had divided from one to six times. The duration of the process of apoptosis once it was initiated was independent of the phase in which the cell was irradiated. Cell death was defined as cessation of movement, typically 20-60 min after the cell rounded with membrane blebbing, but membrane rupture did not occur until 5 to 40 h later. The times to apoptosis and the number of divisions after irradiation were less for cells irradiated late in the cycle. Cells irradiated in G(1) phase divided one to six times and survived 40-120 h before undergoing apoptosis compared to only one to two times and 5-40 h for cells irradiated in G(2) phase. The only cells that died without dividing after irradiation were irradiated in mid to late S phase. Essentially the same results were observed for a dose of 9.5 Gy, although the progeny died sooner and after fewer divisions than after 4 Gy. Regardless of the phase in which they were irradiated, the cells underwent apoptosis from 2 to 150 h after their last division. Therefore, the postmitotic apoptosis did not occur in a predictable or programmed manner, although apoptosis was associated with lengthening of both the generation time and the duration of mitosis immediately prior to the death of the daughter cells. After the non-clonogenic cells divided and yielded progeny entering the first generation after irradiation with 4 Gy, 60% of the progeny either had micronuclei or were sisters of cells that had micronuclei, compared to none of the progeny of clonogenic cells having micronuclei in generation 1. However, another 20% of the non-clonogenic cells had progeny with micronuclei appearing first in generation 2 or 3. As a result, 80% of the non-clonogenic cells had progeny with micronuclei. Furthermore, cells with micronuclei were more likely to die during the generation in which the micronuclei were observed than cells not having micronuclei. Also, micronuclei were occasionally observed in the progeny from clonogenic cells in later generations at about the same time that lethal sectoring was observed. Thus cell death was associated with formation of micronuclei. Most importantly, cells irradiated in late S or G(2) phase were more radiosensitive than cells irradiated in G(1) phase for both loss of clonogenic survival and the time of death and number of divisions completed after irradiation. Finally, the cumulative percentage of apoptosis scored in whole populations of asynchronous or synchronous populations, without distinguishing between the progeny of individually irradiated cells, underestimates the true amount of apoptosis that occurs in cells that undergo postmitotic apoptosis after irradiation. Scoring cell death in whole populations of cells gives erroneous results since both clonogenic and non-clonogenic cells are dividing as non-clonogenic cells are undergoing apoptosis over a period of many days.