ToxSci Advance Access originally published online on February 27, 2007
Toxicological Sciences 2007 97(1):120-127; doi:10.1093/toxsci/kfm032
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Induction of Micronuclei by Phenol in the Mouse Bone Marrow: I. Association with Chemically Induced Hypothermia
The Dow Chemical Company, Toxicology and Environmental Research and Consulting, Midland, Michigan 48674
1 To whom correspondence should be addressed. Fax: 517-638-9863. E-mail: pjspencer{at}dow.com.
Received November 7, 2006; accepted February 21, 2007
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ABSTRACT |
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Doses of xenobiotics at or near LD50 may result in substantial hypothermia in mice. Hypothermia has previously been associated with an increase in micronuclei (MN) formation. The present series of investigations examined the potential for phenol to induce hypothermia in mice and its correlation to previously reported MN induction. In order to examine the potential etiology of phenol-induced MN, evaluation of kinetochore status of MN was also carried out. Phenol-induced hypothermia was assessed in CD1 mice following a single ip dose of phenol ranging from 0–500 mg/kg. Phenol at 300 mg/kg or above caused significant and prolonged hypothermia in male and female mice (up to 7°C decrease). In the micronucleus test, single ip doses of phenol to CD1 mice at 0, 30, 100, or 300 mg/kg produced a significant and prolonged hypothermia and a significant increase in MN only at 300 mg/kg; no marked effect on either body temperature or MN was observed at lower doses. A statistically significant increase in kinetochore-positive MN was observed at the 300-mg/kg dose; however, the response was considerably less than that observed for a known spindle poison. Hence, the induction of MN by phenol occurred only at a dose that produced substantial and prolonged physiologic hypothermia, but interruption of the cell spindle apparatus appeared to play only a minor role in MN formation. These data are suggestive of a threshold mechanism for the induction of MN by phenol treatment in mice.
Key Words: Hypothermia; mouse micronucleus test; phenol; kinetochore.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Phenol (CAS no. 108-95-2), a white crystalline material, is used in various industrial applications including the production of bisphenol A and caprolactam, phenolic and epoxy resins, herbicides and wood preservatives, and for the extraction of lubricating oil from residual oil in the petroleum industry. It is also used in some consumer applications such as throat lozenges, medical disinfectants, ointments, and facial skin peels. With rare exceptions, human exposure in industry has been limited to accidental contact with skin or by vapor inhalation; oral or dermal exposure at very low doses may also occur in humans as a result of its use in medicinal or cosmetic applications.
Phenol is acutely toxic with oral LD50s for mice reported to be 285 mg/kg (95% confidence limits of 256–310) (IUCLID, 2003) and ip LD50s for mice reported to be 180 mg/kg (RTECS, 2004). The acute toxicity of phenol is manifested as muscular tremors of the head (twitching) which spreads to other regions of the body, progressing to convulsions and alterations in autonomic, neuromuscular, and sensorimotor function.
In vitro and in vivo assays for genotoxicity have shown mixed results for phenol with the preponderance of evidence supporting only a weak genotoxic action. These effects are best explained by an understanding of phenol's metabolism by oxidative and conjugation pathways since at doses saturating its conjugative metabolism, oxidation to hydroquinone and other reactive oxidation products has been demonstrated (Koster et al., 1981
).
A reevaluation of the gene mutation studies in bacteria (Epler et al., 1979; Florin et al., 1980; Haworth et al., 1983; Pool and Lin, 1982; Rapson et al., 1980) and gene mutation studies in vitro (i.e., mouse lymphoma) (McGregor et al., 1988; Wangenheim and Bolcsfoldi, 1988) shows that these data would not be considered positive for mutagenicity when evaluated using current criteria (Moore et al., 2003). In vitro assays examining sister chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells have reported small increases in SCEs mostly in the presence of significant cell cycle delay (Ivett et al., 1989; Miller et al., 1995). Increases in SCEs and/or total chromosomal aberrations have also been reported in cultured human lymphocytes (Erexson et al., 1985; Jansson et al., 1986; Morimoto et al., 1983).
Extensive in vivo studies to examine the potential of phenol to induce micronuclei (MN) in the mouse micronucleus test (MNT) have been conducted (Barale et al., 1990; Chen and Eastmond, 1995; Ciranni et al., 1988a,b; Gad-El Karim et al., 1986; Gocke et al., 1981; Mavournin et al., 1990; Marrazzini et al., 1994; McFee et al., 1991; Shelby et al., 1993). In these studies, only where the ip treatment of mice with phenol reached doses that approached or exceeded the LD50 was a weak positive response observed (Chen and Eastmond, 1995; Ciranni et al., 1988a; McFee et al., 1991; Shelby et al., 1993). A limited number of oral studies reported similar results in the bone marrow of mice, again only at near-lethal dose levels (Ciranni et al., 1988a,b). Phenol treatment did not induce an increase in MN at sublethal doses by either route of administration.
Phenol was negative for carcinogenicity in bioassays conducted in two species (NCI, 1980). Phenol belongs to a subset of compounds negative in oncogenicity studies but eliciting a weak positive response in the MNT; the other similar compounds include ascorbic acid, titanium dioxide, and 2,6-diaminotoluene (Shelby et al., 1993). These agents may elicit their weak effects in the MNT via secondary mechanisms for MN formation, rather than a direct interaction with the genetic material.
Of particular interest in the context of a secondary mechanism is the hypothesis that phenol at toxic doses may induce hypothermia in mammals (Allan, 1994). If high doses of phenol induce hypothermia in the mouse, it is conceivable that MN formation is a secondary consequence of this physiologic change since Asanami et al. (1998) and Asanami and Shimono (1997) have shown an association between transient physiologic hypothermia induced by the psychoactive drug chlorpromazine and the formation of MN in the mouse MNT. The induction of MN by transient physiologic hypothermia was hypothesized to be linked to a body temperature (BT)–sensitive impairment of microtubule assembly during mitosis and therefore is expected to be of a threshold nature. That is, no increase in MN formation is expected to occur at doses below that needed to produce physiologic hypothermia. In contrast, the direct interaction of xenobiotics (or their metabolites) with DNA and/or chromatin to produce clastogenesis is generally not considered to be a threshold phenomena. Thus, from a hazard identification perspective, the important question is whether the weak effects observed by previous investigators at near-lethal doses of phenol were directly related to the interaction of phenol and/or its metabolite with the chromosomes (that is, a direct genotoxic effect) or a secondary consequence of phenol-induced hypothermia or both.
The objective of this study was to examine whether phenol administered to mice at high ip doses was capable of causing significant physiologic hypothermia and to assess the association of any phenol-induced changes in BT to the induction of MN in the mouse MNT. The presence of kinetochores in the MN from phenol-treated mice was also assessed to establish if any observed increase in MN was a result of interference with the cell spindle apparatus, the mechanism postulated for hypothermia. The kinetochore is a centromere-associated protein. MN positive for kinetochores generally result from spindle disturbances during erythropoiesis, and it is assumed that a kinetochore-positive MN contains an entire chromosome. Thus, scoring MN for the presence of kinetochores is a method to test for aneuploidy-inducing agents.
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MATERIALS AND METHODS |
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Chemical Substances
Phenol (CAS no. 108-95-2; purity > 99%) was obtained from Harrell Industries, Rock Hill, South Carolina; cyclophosphamide monohydrate (CP) (CAS no. 6055-19-2) and vinblastine (VB) (CAS no. 143-57-9) were obtained from Sigma Chemical Co. (St Louis, MO).
Animals and Treatment
Eight week-old male and female outbred CD1(1CR)BR (referred to as CD1) mice were obtained from Charles River Laboratories (Portage, MI). Mice were housed individually in stainless steel cages, in rooms designed to maintain adequate environmental conditions (temperature, humidity, and photocycle). Mice were fed Purina Certified Rodent Chow #5002 (Purina Mills, Inc., St Louis, MO) and municipal drinking water was provided ad libitum. After a 7-day acclimation period, mice were randomly assigned to control or phenol-treated groups. Phenol and the vehicle control, phosphate-buffered saline (PBS) and VB (4 mg/kg; positive control for kinetochore-positive MN), were administered by a single ip injection. The ip route of administration was selected for phenol because this route was used in studies previously reporting positive results in the MN test. CP was administered by single oral gavage (120 mg/kg). The routes of administration for positive control substances were consistent with the laboratories historical positive control database. The dosing volume for oral and ip administrations was 10 ml/kg of body weight. Animals were housed in a facility approved by the American Assessment and Accreditation of Laboratory Animal Care International. The Institutional Animal Care and Use Committee approved the study. All study phases were compliant with Good Laboratory Practice Standards (USEPA, 1989).
Overview of Study Design
The current investigation was carried out in two phases. The objective of phase I (In "Hypothermia Experiment" section) was to establish the maximum tolerated dose (MTD) in mice for a single ip injection of phenol and to assess the potential for phenol to induce hypothermia. In phase II (In "Mouse MNT" section), the objective was to determine the relationship between phenol-induced hypothermia and the induction of MN and to examine the etiology of MN by evaluating the kinetochore status of MN as a measure of spindle disturbances during cell divisions.
Hypothermia Experiment
Four mice per sex received a single ip dose of the vehicle control (PBS) or 50, 150, 200, 300, 400, or 500 mg/kg phenol. The relative BTs of the animals were monitored sc using programmable transponders that also served for animal identification (BioMedic Data Systems, Seaford, DE). The temperatures were collected by scanning the microchip immediately prior to dosing and at approximately 5, 30, 60, and 90 min and 2, 3, 4, 5, 6, 24, and 48 h after dosing. Clinical signs of toxicity (e.g., phenol twitching behavior, lethargy, morbidity, mortality) were evaluated in each mouse at the same time points as BT.
MNT and Kinetochore Experiments
Study design.
The MNT and kinetochore evaluation were conducted in two successive independent experiments. For the MNT groups of mice (6/sex/dose/killing time) received a single ip injection of 0 (vehicle control), 30, 100, or 300 mg/kg phenol. Another group of six mice per sex received a single 120 mg/kg dose of CP by oral gavage to serve as the positive control for MN formation. BT and clinical observations of animals in all dose groups were monitored using the procedures previously described, just prior to dosing and at 2, 5, 24, and 48 h after dosing. Animals were killed either 24 or 48 h after dosing; a bone marrow sample was collected from femurs immediately after killing from all surviving animals. Positive control animals were killed only at one time point, that is, 24 h after dosing. For the kinetorchore, evaluation groups of male mice (6/dose/sacrifice time) received a single ip injection of 0 (vehicle control) or 300 mg/kg phenol. A single phenol dose group was evaluated since 300 mg/kg phenol was the only dose to cause an increased frequency in MN in the initial MNT study. Another group of six mice/sex received a single 120 mg/kg dose of CP by oral gavage to serve as the positive control for MN formation or VB, 4 mg/kg, positive control for kinetochore-positive MN administered by a single ip injection and 5 h after dosing. Animals were killed 24 h after dosing. For both experiments, a bone marrow sample was collected from femurs immediately after sacrifice from all surviving animals; positive control animals were sacrificed only at one time point, that is, 24 h after dosing.
Slide preparation.
All mice were euthanized via carbon dioxide inhalation in a closed chamber environment. The bone marrow was removed from both femurs by aspiration into fetal calf serum and centrifuged at approximately 1000 rpm for 5 min. The cell pellet was resuspended in a drop of serum and a wedge film prepared on a microscope slide. The slides were allowed to dry prior to staining with Wright-Giemsa using an automatic slide stainer (Ames Hema-Tek, Miles Scientific, Naperville, IL). Slides for kinetochore analysis were air dried and stored for subsequent staining.
Slide staining for kinetochores.
Slides stained for kinetochore-positive MN were evaluated for control, 300 mg/kg phenol, and VB groups. The kinetochore staining consisted of attaching and stacking a series of antibodies to the kinetochores of chromosomes (Eastmond, personal communication). Prior to adding the antibodies, the slides were fixed in 2% paraformaldehyde, rinsed with PBS and soaked in PBS + 0.01% Tween 20 (PBST; Sigma). Slides were treated with a sodium phosphate buffer containing 0.1% Triton X-100 and milk proteins (PTM) to block nonspecific antibody binding. The centromere-positive control serum (Antibodies Inc., Davis, CA) was incubated on the slides at 37°C for 1.25 h in a humidified chamber. Following incubation, the slides were removed from the humidified chamber and rinsed with PBST. Anti-human IgG fluorescein isothiocyanate (FITC) antibodies (CHEMICON, Temecula, CA) were incubated on the slides at 37°C for 1.25 h in a humidified chamber. Following the second incubation, the slides were removed from the humidified chamber and were rinsed with PBS, PBST, and PTM. The fluorescent signal was amplified using a third antibody, rabbit anti-goat IgG FITC (Antibodies Inc., Davis, CA), incubated on the slides for 1.25 h. At the end of the third incubation, the slides were removed from the humidified chamber and rinsed with PBS. The slides were then stained with propidium iodide (1–5 µl) for 1–2 min, rinsed with PBS and mounted with 100–200 µg/ml of phenylenediamine in glycerol (Sigma).
Slide scoring—MN-PCE.
The slides were coded, scored blindly to control for bias, and decoded upon completion. Two thousand polychromatic erythrocytes were examined from each animal and the number of micronucleated polychromatic erythrocytes (MN-PCE) was recorded. In order to determine the ratio of PCE to normochromatic erythrocytes (NCE) in the bone marrow, approximately 200 erythrocytes from each animal were examined and the ratio was expressed as percentages: (PCE x 100/PCE + NCE).
Kinetochores in MN-PCE.
The anti-kinetochore–stained slides were coded prior to the evaluation of MN with kinetochores. Whenever possible, up to 20 MN were analyzed for the occurrence of kinetochores. A fluorescent microscope equipped with a blue excitation filter (460–490 nm) combined with a suppression filter of 520 nm was used to analyze these slides. With propidium iodide counterstaining, the MN emitted a right red color. If a kinetochore was present, a bright yellow dot was observed within the MN.
Statistics.
The raw data on the counts of MN-PCE for each animal were first transformed by adding one to each count and then taking the natural log of the adjusted number. The transformed MN-PCE data and the data on percent PCE were analyzed separately by a three-way analysis of variance (both sexes were used in the study) (Winer, 1971). The sex by dose by time interaction in the three-way analysis of variance was considered to be zero. From this initial analysis, the two-way interactions were reviewed for significance. Based upon the review, the data were analyzed by one, two, or three-way analysis looking only at main effects. Pairwise comparisons of treated versus control groups were done, if the dose effect was significant, by Dunnett t-test, one sided (upper) for MN-PCE and two sided for the percent PCE (Winer, 1971). Linear dose-related trend tests were performed if any of the pairwise comparisons yield significant differences. Kinetochore-positive MN-PCEs were compared using Fisher exact test. The alpha level at which all the test data were conducted was 0.05.
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RESULTS |
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Hypothermia Experiment
Mortality/clinical signs.
All mice dosed with 400 or 500 mg/kg phenol died within 24 h after dosing. A single male and a single female mouse in the 300-mg/kg dose group died prior to the end of the 48-h observation period (Table 1). No deaths occurred in phenol-treated mice at dose levels of 200 mg/kg and lower. Clinical signs of toxicity which were sometimes severe, included reduced activity, twitching, and tremors, and were observed shortly after dosing in mice treated with 100 mg/kg of phenol and higher, although decrease activity was observed only at doses of 200 mg/kg and above. Surviving mice appeared normal approximately 1 h after dosing and exhibited no further clinical signs of toxicity. Qualitatively, males appeared to be somewhat more sensitive to the lethal doses of phenol as indicated by a greater incidence of tremors/convulsions occurring only minutes after dosing and by a shorter time to death (first death 2 h after dosing) as compared to female mice (first death 6 h after dosing; Fig. 1).
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400 mg/kg caused 100% lethality by 24 h after dosing.Body temperature.
Marked, prolonged and a dose-dependent BT depression was observed in male and female mice receiving a single ip injection of 300 mg/kg phenol and higher (Fig. 1). In the 300-mg/kg dose group, the mean predosing BT was 36.7°C and 37.0°C in male and female mice, respectively. Thirty minutes after dosing, mean BTs decreased to approximately 32°C with mean BT as low as 28°C at 5 h after dosing in both sexes. BT in this group of mice did not return to baseline within 48 h and remained depressed 4–5°C at the conclusion of the experiment. For the animals that survived until 48 h at the three highest doses of phenol, BT decrements up to 8°C from mean predosing BTs were recorded in both sexes. A smaller and transient decrease in BT occurred in mice treated with 100, 150, or 200 mg/kg phenol. Up to 4°C decrease occurred at the 200-mg/kg dose beginning at 1 h after dosing and returned to values within the range of control data by 4 h after dosing at all doses (Fig. 1). Smaller and more transient changes in BT were observed at doses of 100 and 150 mg/kg, with no changes in BT observed in mice following treatment with a 50-mg/kg dose of phenol.
Mouse MNT
Mortality/clinical signs.
A single death, not attributed to treatment with the test article, occurred during the conduct of the mouse MNT in one male mouse dosed with 30 mg/kg phenol. All other mice survived to the end of the study. About one third of the male mice and about one half of the female mice dosed with 300 mg/kg phenol showed typical phenol-induced clinical signs (i.e., twitching/tremors) consistent with those observed in the hypothermia study (Table 1). At this dose level, clinical signs of toxicity developed within minutes after dosing and persisted for approximately 1 h. Mice dosed with 100 mg/kg phenol exhibited twitching/tremors immediately after dosing; however, symptoms at this dose level persisted for only several minutes and subsided before the 1-h clinical observation. No treatment-related clinical signs were observed in mice receiving a 30-mg/kg dose of phenol.
Body temperature.
Mice dosed with 300 mg/kg phenol exhibited a substantial and prolonged hypothermia that was consistent with the effects on BT observed in the hypothermia study (data not shown). In the 300-mg/kg dose group the mean pre-dosing BT was 35.7 and 36.3°C in male and female mice, respectively. Twenty-four hours after dosing mean BTs decreased to approximately 31.5°C in both sexes. By 48 h after dosing, the degree of BT decrement was approximately 7°C (28.6°C) in male mice and 6°C (30.1°C) in female mice. BT was unaffected by phenol treatment at the lower doses of 100 and 30 mg/kg in both male and female mice (Fig. 2). BT of mice treated with the positive control (120 mg/kg CP) was unaffected (Table 2).
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MN test.
Male and female mice treated with phenol at a dose of 300 mg/kg had statistically significant increases in the frequency of MN-PCE at 24 and 48 h after dosing (Table 2, Fig. 2). The mean frequency of MN-PCE/1000 PCE at 24 h for control and high-dose males was 2.1 and 10.8, respectively and for females, this value was 2.5 and 11.3, respectively (Table 2). At 48 h, the frequency of MN-PCE in mice treated with phenol at 300 mg/kg increased over that observed at 24 h with the same dose; the mean frequency of MN-PCE/1000 PCE was 18.3 and 17.8 for males and females, respectively. Overall, the increased frequency in MN-PCE at both 24 and 48 h was only observed in the presence of marked hypothermia which was observed in the highest dose group only.
The mean percent PCE values were reduced at 24 h (all dose levels of phenol, both sexes) and at 48 h for the group treated with phenol at 300 mg/kg (both sexes), indicating significant effect of the treatment on erythropoiesis. There were no significant differences from controls in the frequency of MN-PCE or in the mean percent PCE (48 h) for mice treated with doses of phenol lower than 300 mg/kg.
Mice treated with the positive control, cyclophosphamide, showed a significant increase in the frequency of MN-PCE as compared to the negative controls. Percent PCE values of the positive control animals were found to be significantly lower than those of the negative control animal (Table 2).
Kinetochore experiment.
Similar increases in the frequency of MN were observed in this experiment as in the first MNT study. A statistically significant increase in the proportion of kinetochore-positive (K+) MN was observed in phenol mice compared to the negative controls (Table 3). The positive control, VB, induced a significant increase in (K+) MN consistent with its mode of action on spindle assembly. The proportion of (K+) MN induced by VB was substantially greater than that observed for phenol (phenol (K+) MN = 13%; VB (K+) MN = 78%).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The current series of investigations demonstrated the ability of high doses of phenol, administered to mice as a single ip dose, to cause marked and prolonged decreases in BTs. Additionally, increases in the frequency of MN-PCE were noted only in the presence of marked hypothermia. Phenol at doses of 300 mg/kg or greater induced, on average, a 5–7°C drop in BT over a 48-h observation period. Notably, doses of phenol-inducing hypothermia were commonly associated with phenol twitching behavior and substantial mortality (100% at 400 or 500 mg/kg and 20% at 300 mg/kg phenol). Phenol twitching was also noted in mice dosed with 100 mg/kg phenol in the absence of a marked depression in BT; however, clinical symptoms were very brief, subsiding within minutes after dosing. Clinical signs coupled with deaths in mice receiving 300 mg/kg phenol indicated that 300 mg/kg ip was a MTD for conducting a MNT in this strain of mice. A novel finding is that only doses of phenol at or above the MTD were capable of producing significant and prolonged hypothermia.
Hypothermia has been studied as an adaptive effect in rodents that modulates the acute lethality for some toxicants (Gordon, 1991, 2005; Gordon and Watkinson, 1988; Gordon et al., 1988; Watkinson and Gordon, 1993; Watkinson et al., 1989). It is now well documented that a physiologic response in some rodents in response to exposure to toxic agents is a lowering of their core BT which may attenuate the toxic response and increase survival (Watkinson and Gordon, 1993). In the current study, only acutely toxic doses of phenol (i.e., doses producing some lethality) were associated with marked prolonged hypothermia. Hypothermia as a physiological response to toxicants is apparently different between rodents and humans (Gordon, 1991). Humans do not respond to toxic insults by downregulating BT as do rodents which brings into question the relevance of toxicity data, including genotoxicity, generated in rodents at high, toxic doses and its utility for predicting acute human hazards.
Only recently has hypothermia been suggested as a cause of increased MN formation as a result of observing that temperature changes alone in cultured CHO cells and chemically-induced transient changes in the BT in mice were associated with an increased frequency of MN (Asanami and Shimono, 1997; Asanami et al., 1998, 2001). Similarly, in the current investigations, increases in MN formation were exclusively associated with doses of phenol producing marked prolonged physiologic hypothermia. Statistically significant increases in the frequency of MN-PCE were observed in 24 and 48 h bone marrow smears of mice dosed with 300 mg/kg phenol. The mean percent PCE values were significantly reduced in this treatment group indicating some phenol-induced toxicity to the bone marrow. Mice receiving sublethal doses of phenol (30 or 100 mg/kg) were not hypothermic and had no increased frequency of MN-PCE at either harvest time point even though moderate phenol toxicity (as determined by twiches/tremors) was present in mice dosed with 100 mg/kg, indicating that phenol was systemically available to reach the target tissue, bone marrow. These data suggest a threshold for the induction of phenol-induced hypothermia, which is associated with a no observable effect level for MN induction in phenol-treated mice. Thus, results from our investigations suggest the possibility that MN were induced not by phenol directly but rather a secondary effect of the treatment on the regulation of BT.
Confirming the presence of kinetochores in the MN from phenol-treated mice was a method used to assess the orign of MN in phenol-treated mice. Hypothermia-induced genotoxicity was previously speculated to originate from an aneugenic mechanism via disturbance of the mitotic apparatus (Asanami et al., 1998). Specifically, transient changes in core BT were hypothesized to interfere with the polymerization of subunits that form microtubules, primarily at the kinetochores, resulting in a lagging whole chromosome leading to MN formation (Parton et al., 1991). Alternately, it is possible that hypothermia may not induce MN through altering cell spindle mechanisms.
Although a statistically significant increase in the proportion of kinetochore-positive MN was observed in phenol-treated mice relative to the negative control, this increase was substantially less than that observed for the known spindle poison, VB. Thus, while there is some evidence for a mode of action that, at least in part, involves disruption of the spindle apparatus of the cell, the majority of MN induced by phenol apparently originates from clastogenesis through yet unknown mechanisms. In this context, an alternative mechanism that cannot be excluded is the role of the phenol metabolite, hydroquinone. Synergistic increases in MN formation were shown by the coadminstration of hydroquinone and phenol which induced extensive chromosome breakage in the euchromatic regions of the mouse bone marrow (Barale et al, 1990; Chen and Eastmond, 1995). This pattern of genotoxicity appears consistent with the data from the current investigation. Therefore, it is conceivable that very high doses of phenol lead to pharmacokinetic conditions favoring the formation and subsequent interactive effects of phenol and hydroquinone. Nevertheless, MN formation exhibited a dose threshold, which correlated with phenol-induced hypothermia, and thus, the mode of action is likely to be secondary to the induced hypothermia. Overall, these studies suggest a role, but not necessarily a causality, for phenol-induced hypothermia in the formation of MN. Studies to determine MN frequency when hypothermia is induced by physical rather than chemical means and to determine if the maintenance of BT through thermoregulatory support would ameliorate MN formation in phenol-treated animals are logical follow-up studies to determine the role of hypothermia to MN formation (manuscripts in preparation).
The current series of studies established, for the first time, the ability of phenol at doses confirmed to be at or above the MTD, to induce a substantial and prolonged hypothermia in CD mice. Furthermore, significant increases in the frequency of MN were exclusively associated with phenol-induced hypothermia and altered thermoregulatory patterns, which exhibit a clear threshold. Interruption of the cell spindle apparatus appears to play a partial role in MN formation in phenol-treated mice. However, the mechanism by which hypothermia induces MN is not clearly established in the literature. Most importantly, this study raises questions on the relevance of positive results obtained in the MNT in the presence of significant decreases in BT, a protective physiologic adaptation that is unique to rodents and may occur frequently at doses at or above the MTD established for the MNT.
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ACKNOWLEDGMENTS |
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The authors wish to thank American Chemistry Council's Phenol Panel, Arlington, VA, who sponsored the current body of research, Joy Grundy for her help in the conduct of the study, Anne Linscombe for expertise in conducting the kinetochore evaluation, and Lisa McFadden for her assistance with the statistical analyses. Conflicts of interest: The authors acknowledge that they are employed by The Dow Chemical Company, which manufactures and sells phenol.
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