Cardiovascular and Thermoregulatory Effects of Inhaled PM-Associated Transition Metals: A Potential Interaction between Nickel and Vanadium Sulfate

Matthew J. Campen*,1, Julianne P. Nolan{dagger}, Mette C. J. Schladweiler{dagger}, Urmila P. Kodavanti{dagger}, Paul A. Evansky{dagger}, Daniel L. Costa{dagger} and William P. Watkinson{dagger}

* Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, North Carolina 27599; and {dagger} Pulmonary Toxicology Branch, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711

Received April 16, 2001; accepted August 15, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent epidemiological studies have shown an association between daily morbidity and mortality and ambient particulate matter (PM) air pollution. It has been proposed that bioavailable metal constituents of PM are responsible for many of the reported adverse health effects. Studies of instilled residual oil fly ash (ROFA) demonstrated immediate and delayed responses, consisting of bradycardia, hypothermia, and arrhythmogenesis in conscious, unrestrained rats. Further investigation of instilled ROFA-associated transition metals showed that vanadium (V) induced the immediate responses, while nickel (Ni) was responsible for the delayed effects. Furthermore, Ni potentiated the immediate effects caused by V when administered concomitantly. The present study examined the responses to these metals in a whole-body inhalation exposure. To ensure valid dosimetric comparisons with instillation studies, 4 target exposure concentrations ranging from 0.3–2.4 mg/m3 were used to incorporate estimates of total inhalation dose derived using different ventilatory parameters. Rats were implanted with radiotelemetry transmitters to continuously acquire heart rate (HR), core temperature (TCO), and electrocardiographic data throughout the exposure. Animals were exposed to aerosolized Ni, V, or Ni + V for 6 h per day x 4 days, after which serum and bronchoalveolar lavage samples were taken. Even at the highest concentration, V failed to induce any significant change in HR or TCO. Ni caused delayed bradycardia, hypothermia, and arrhythmogenesis at concentrations > 1.2 mg/m3. When combined, Ni and V produced observable delayed effects at 0.5 mg/m3 and potentiated responses at 1.3 mg/m3, greater than were produced by the highest concentration of Ni (2.1 mg/m3) alone. These results indicate a possible synergistic relationship between inhaled Ni and V, and provide insight into potential interactions regarding the toxicity of PM-associated metals.

Key Words: radiotelemetry; hypothermia; particulate matter; temperature; arrhythmia; heart rate; nickel; vanadium; bradycardia; fly ash; inhalation; whole-body.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Epidemiological reports have demonstrated a positive association between increases in particulate matter (PM) air pollution levels and excess daily morbidity and mortality from respiratory and cardiovascular causes (Burnett et al., 1995Go; Ponka and Virtanen, 1996Go; Pope et al., 1992Go; Schwartz and Morris, 1995Go). To determine whether a causal relationship exists between these outcomes, many studies of PM toxicity have been conducted over the past several years. A major focus of this research effort has been to identify which, if any, specific characteristics or constituents of PM mediate the observed toxic effects. Investigations of residual oil fly ash (ROFA), a component of ambient PM in certain regions of the U.S., revealed that specific soluble transition metals, namely iron (Fe), nickel (Ni), and vanadium (V), were responsible for the majority of ROFA toxicity (Dreher et al., 1997Go; Dye et al., 1999Go; Kodavanti et al., 1997Go).

Previous studies of ROFA toxicity administered the particulate material by intratracheal instillation (IT), a method with particular strengths and weaknesses as detailed by Driscoll et al. (2000). Instilled ROFA caused both immediate (0–6 h postinstillation) and delayed (24–96 h postinstillation) physiological responses, including bradycardia, hypothermia, and arrhythmogenesis, in conscious, unrestrained rats (Campen et al., 2000aGo; Watkinson et al., 1998Go). Further investigation of the ROFA-associated transition metals clearly demonstrated that instilled V caused the immediate effects, while instilled Ni was responsible for the delayed response (Campen et al., manuscript in preparation). Moreover, certain interaction effects occurred when these metals were administered in combination, including an apparent synergism between Ni and V and an attenuating effect from Fe. Specifically, Ni exacerbated the immediate response to V in a more than additive manner, while Fe coexposure reduced the effects of both V and Ni + V instillation. It was proposed that the potentiating effect of Ni on the immediate V response with no discernible interaction effect on the delayed response to Ni was a possible artifact of the bolus nature of the instillation exposure methodology. That is, given its short pulmonary residence time (~85% of VSO4 is cleared by 3 h post-IT), most of the highly soluble V would have been cleared by the time Ni began to affect the lung (Rhoads and Sanders, 1985Go). However, metal exposures during a prolonged air pollution episode would be concomitant, and it is reasonable to expect that the delayed effects of Ni could be compounded by the presence of V in such situations.

ROFA inhalation studies have been conducted with inconsistent results. Killingsworth et al. (1997) exposed rats with monocrotaline (MCT)-induced pulmonary hypertension and vasculitis to 0.6 mg/m3 ROFA and found increased cytokine gene expression in the heart and lung tissue, along with increased mortality. However, Kodavanti et al. (1999) reported no mortality in MCT-treated rats following nose-only exposure to 15 mg/m3 ROFA (25x that of Killingsworth et al., 1997Go), and no heart rate (HR) or core temperature (TCO) alterations during exposures. ROFA-exposed rats did display a greater degree of bradycardia than did filtered air-exposed rats beginning after the third day of exposures, although both groups showed adverse health effects related to the MCT toxicity (Watkinson et al., 2000Go). In a similar study, electrocardiographic ST segment depression, a sign of possible cardiac ischemia, was noted in spontaneously hypertensive rats exposed to 15 mg/m3 ROFA, but again no lethalities occurred (Kodavanti et al., 2000Go).

The purpose of the present study was to corroborate the findings of both transition metal IT studies and ROFA inhalation studies, and also to determine if concomitant exposure to Ni and V would lead to a synergistic potentiation of the immediate and delayed responses observed in IT studies. Because ventilation and uptake of particles is difficult to accurately measure or predict, several concentrations of metals were used to ensure that the results from the present study could be compared to those obtained from the instillation studies. In addition to the physiological parameters (HR, ECG, and TCO), blood samples were analyzed for fibrinogen and cell content and the lungs were lavaged to assay for biomarkers of pulmonary injury and inflammation. While previous studies have used the MCT model of pulmonary disease, only healthy adult rats were used in the present study to ensure greater homogeneity of response.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Male Sprague-Dawley rats (Charles River Laboratory, Raleigh, NC; 60 days old at the beginning of study) were used in all protocols. Rats were quarantined for 1 week following delivery to ensure that all animals were pathogen-free and to allow recovery of normal circadian rhythm. Following telemeter implantation, rats were individually housed in Plexiglas cages (28 x 17 x 12 cm) within a specially designed, climate-controlled chamber. All rats were moved to the exposure chambers 72 h prior to initiation of exposures to allow acclimation. Within the exposure chamber, animals remained in specially modified metal cages with one Plexiglas wall designed to optimize the reception of radiotelemetry signals emitted from the rats. The ambient temperature in all chambers was controlled at 22 ± 1°C and the relative humidity was maintained from 40–65%. A 12-h light:12-h dark cycle was imposed with food and water provided ad libitum throughout the experiment. Rats remained in these chambers throughout the exposure period, but were returned to the original housing chamber immediately following the final day of exposure. All protocols were approved by the Institutional Animal Care and Use Committee of the U.S. EPA.

Surgical preparation.
Animals were anesthetized with pentobarbital sodium (Nembutal; Abbott Laboratories, North Chicago, IL; 50 mg/kg, ip) and implanted with a biopotential radiotelemetry transmitter (Model TA11CTA-F40; Data Sciences International, Inc., St. Paul, MN) using aseptic surgical procedures (Watkinson et al., 1995Go). The body of the telemeter was placed within the peritoneal cavity to allow measurement of TCO, and biopotential leads were sutured subcutaneously on either side of the thoracic cavity to obtain an ECG signal similar to that of Lead II from the standard ECG. Animals were allowed 7 days for recovery from surgery and to reestablish circadian rhythm. At the time of metals exposure, body weights of all rats averaged 390 g, with a SD of 35 g.

Experimental protocol.
Groups of rats (n = 4 per group) were exposed to various concentrations of individual metals 6 h per day for 4 days and monitored for 96 h following the final exposure, according to the protocol detailed in Table 1Go. In addition to the telemetered rats, an equal number of naïve (untelemetered) rats of the same strain and age were exposed simultaneously and sacrificed 24 h following the final exposure. At the completion of the 24- or 96-h postexposure monitoring period, each rat was anesthetized with pentobarbital and euthanized via exsanguination and pneumothorax. Lung, blood, and cardiac tissues were collected from all animals.


View this table:
[in this window]
[in a new window]
 
TABLE 1 Summary of Protocols for Inhaled Transition Metals Study
 
The lungs were lavaged using methods previously described (Kodavanti et al., 1999Go). Briefly, right lung lobes (~ 60% lung volume) were lavaged using Ca2+/Mg2+-free phosphate-buffered saline (pH 7.4) at a volume of 16.8 ml/kg body weight. Total cell counts were determined using a Coulter counter (Coulter, Inc., Miami, FL) and differential cell counts were determined from a second aliquot of bronchoalveolar lavage fluid (BALF) using a Shandon 3 Cytospin (Shandon). The remaining BALF was centrifuged (1500 x g for 10 min) to remove cells. Assays for N-acetyl glucosamide (NAG) activity, microalbumin (MIA), and lactate dehydrogenase (LDH) activity were performed on the remaining BALF by a Hoffman-LaRoche Cobas Fara II clinical analyzer (Roche Diagnostics, Branchburg, NJ). Total protein levels were determined using a Coomassie Plus protein assay kit (Pierce, Rockford, IL). Blood was analyzed for CBC and fibrinogen levels using standard clinical laboratory techniques (Gardner et al., 2000Go).

Estimation of equivalent exposures.
The doses used in the original metal instillation study (263 µg NiSO4 · 6H2O, and 245 µg VSO4) were designed to match the content of each metal in 2.5 mg of ROFA, the highest dose used in the study by Campen et al. (2000a). Because the effects of instilled ROFA and Ni lasted 84–96 h, the inhalation exposures of the present study were conducted over 4 days to maximize the difference between the exposure profiles for instillation and inhalation methodologies (i.e., bolus vs. continuous exposure). The inhalation exposure concentrations (C) required over a 4-day exposure to equal the doses (D) from instillation studies were calculated using the following equation:

where is the minute volume, t is duration of exposure (1440 min), and Df is the deposition fraction. For the purposes of this study, was estimated to be between 190 and 300 ml/min (Costa and Tepper, 1988Go), and the Df was estimated as 0.10 for 1 µm particles. Therefore, based on the central and extreme estimates for , the following target concentrations were used for each metal: 0.3, 0.6, 1.2, and 2.4 mg/m3. The second highest concentration (1.2 mg/m3) is the central estimate that most accurately models the dose from the metals IT study (Campen et al., manuscript in preparation).

Aerosolization of metals.
Metals were mixed daily in aqueous solution just prior to exposures. The solution was aerosolized by a 6-jet atomizer (model 9306, TSI) using one jet at 30 psi, 7 l/min. All output was directed to a drying/dilution vessel, passed through a charge neutralizer, and directed into the chambers. Chamber airflow ranged between 90 and 100 l/min. Metal concentration in each chamber was determined by gravimetric analysis. Conditioned teflon filters (47 mm x 1 µm pore, 6 samples per exposure day) were weighed before and after sampling, and the weight difference was divided by the volume of sampled air. Particle size and distribution were determined using a 7- or 8-stage cascade impactor. Temperature and humidity were continuously monitored.

Telemetry data acquisition and analysis.
High speed (50 mm/s x 10 s) chart recordings (Model MT95K2; AstroMed, West Warwick, RI) of the telemetric ECG signals from all animals were acquired at 4-h intervals for 72 h prior to metals exposure. ECG rhythm strips were also obtained at 15-min intervals during the exposure periods and at 1-h intervals during nonexposure periods. Arrhythmias were manually counted from the chart records and divided by the total time of sampling to determine frequency. HR and TCO data for all rats were collected by a computerized acquisition system (Dataquest IV; Data Sciences International Inc., St Paul, MN) at 5-min intervals, from 72 h before until 96 h following the exposures, and stored on disk.

Statistics.
HR and TCO data were averaged within dose groups and analyzed by Tukey's fixed effects ANOVA. All terminal endpoints were averaged by exposure group and compared by ANOVA. Arrhythmia frequency was analyzed using a t-test assuming unequal variances. Probability values of < 0.05 were considered significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Particle Concentration and Distribution
As shown in Table 1Go, the target concentrations were not always attained, although the range of concentrations used was broad enough to ensure comparison of the present results with those of instillation studies. Fortuitously, the Ni and Ni + V concentrations were identical, simplifying comparisons between these exposure groups. Particle sizes averaged 0.65 µm with a geometric standard deviation of 2.11. A chamber distribution study was conducted using aerosolized saline. A positional concentration variation of approximately 20% was observed (Table 2Go). Importantly, the corner cages within the chamber (where telemetried rats were positioned) had consistently lower concentrations (~ 20%) than were found in the center of the chamber where exposure samples were obtained.


View this table:
[in this window]
[in a new window]
 
TABLE 2 Chamber Particle Distribution
 
Vanadium
The low concentrations (0.3–0.9 mg/m3) of V caused no changes in HR or TCO. Control animals demonstrated a robust circadian oscillation of both HR and TCO; this circadian behavior was not disturbed by V exposure (Figs. 1A and 2AGoGo). Exposure to even the highest concentration of V (1.7 mg/m3) caused only the slightest bradycardia ({downarrow}20 bpm; Fig. 1AGo) and hypothermia ({downarrow}0.2°C; Fig. 2AGo), but no increase in arrhythmia frequency (Fig. 3AGo). All such physiologic effects were resolved prior to cessation of the 6-h exposure period. However, BALF endpoints indicate a modest dose-related increase in lung injury and inflammation at both 24-h and 96-h postexposure timepoints (Tables 3 and 4GoGo). Inflammatory cells were increased only in the highest concentration. Few hematological effects were observed following V exposure. Fibrinogen concentrations remained at control levels (Table 3Go), and no significant alterations in the composition of circulatory cell populations were observed following exposure to any metal (data not shown).



View larger version (81K):
[in this window]
[in a new window]
 
FIG. 1. Changes in heart rate for conscious, unrestrained, adult male Sprague-Dawley rats before, during, and after whole-body inhalation of vanadium (A), nickel (B), and combined vanadium and nickel (C). Vertical gray bars indicate periods of exposure (6 h/day x 4 days). Data were collected at 10-min intervals and averaged to 30-min time points for presentation purposes. Also for clarity, data for the lowest 2 concentrations of V (0.3, 0.6 mg/m3) and the lowest concentration of Ni (0.37 mg/m3) are omitted, as no HR effect was observed for these levels.

 


View larger version (74K):
[in this window]
[in a new window]
 
FIG. 2. Changes in core body temperature for conscious, unrestrained, adult male Sprague-Dawley rats before, during, and after whole-body inhalation of vanadium (A), nickel (B), and combined vanadium and nickel (C). Vertical gray bars indicate periods of exposure (6 h/day x 4 days). Data were collected at 10-min intervals and averaged to 30-min time points for presentation purposes. Also for clarity, data for the lowest 2 concentrations of V (0.3, 0.6 mg/m3) and the lowest concentration of Ni (0.37 mg/m3) are omitted, as no TCO effect was observed for these levels.

 


View larger version (66K):
[in this window]
[in a new window]
 
FIG. 3. Changes in arrhythmia frequency for conscious, unrestrained, adult male Sprague-Dawley rats resulting from whole-body inhalation of vanadium (A), nickel (B), and combined vanadium and nickel (C). Arrhythmias were manually counted from chart records obtained before and during exposures, and divided by sampling time to determine the frequencies. Note the increased (5x) y-axis range for the Ni + V exposure (C). The types of arrhythmias observed are similar to those of previous studies (Watkinson et al., 1998Go).

 

View this table:
[in this window]
[in a new window]
 
TABLE 3 Markers of Pulmonary Injury and Plasma Fibrinogen Levels
 

View this table:
[in this window]
[in a new window]
 
TABLE 4 Changes in Lavageable Inflammatory Cell Population in Lungs of Vanadium-, Nickel-, and Vanadium + Nickel-Exposed Rats
 
Nickel
Nickel inhalation led to severe delayed bradycardia, hypothermia, and arrhythmogenesis at the higher concentrations (1.3, 2.1 mg/m3), while the lower concentrations (0.36, 0.49 mg/m3) caused no significant effects on these parameters. Inhalation of 1.3 mg/m3 Ni led to a maximal HR decrease of 75 bpm (Fig. 1BGo) and a TCO decrease of 2.0°C (Fig. 2BGo). These responses began on the third day of exposures and lasted over 72 h. The highest concentration (2.1 mg/m3) caused similar, but exacerbated responses compared to those of the 1.3 mg/m3 concentration. Rats exposed to the highest concentration displayed marked bradycardia and hypothermia ({downarrow}100 bpm and {downarrow}3.3°C, respectively) that began on the second day of exposures and persisted for 96 h. Slight HR increases were observed 3 to 4 days after the end of exposures in all Ni-exposed rats. Arrhythmogenesis was significantly elevated in rats exposed to 1.3 and 2.1 mg/m3 (Fig. 3BGo), beginning on the third day of exposure in the 1.3 mg/m3-exposed rats, and on the second day in the 2.1 mg/m3-exposed rats. Commonly observed arrhythmias included atrioventricular node block and premature depolarizations (see Watkinson et al., 1998Go). Ni exposure caused a dose-related increase in all markers of pulmonary injury and inflammation; these increases were far greater than those observed in V-exposed rats (Tables 3 and 4GoGo). Ni also led to significant dose-related increases in plasma fibrinogen at 24 h; fibrinogen was only elevated in the 2.1 mg/m3 concentration group at 96 h (Table 3Go).

Combined Nickel and Vanadium
Only 2 concentrations of combined Ni + V were examined, as the highest dose of Ni caused profound toxicity and the 0.3 and 0.6 mg/m3 levels of both V and Ni caused no hypothermia or bradycardia. It was decided that combinations at the no-effects level (0.6 mg/m3) and at the low-effects level (1.2 mg/m3) would prove most informative; however, measurements during exposures indicated that rats were actually exposed to 0.5 and 1.3 mg/m3 of both Ni and V. Rats exposed to 0.5 mg/m3 of both Ni and V displayed bradycardic and hypothermic effects ({downarrow}50 bpm and {downarrow}1.0°C, respectively) that began after the third exposure day and persisted just over 30 h (Figs. 1C and 2CGoGo). Arrhythmogenesis was markedly increased on the second and third days of exposure (Fig. 3CGo; note larger scale on y-axis). BALF endpoints (LDH, protein, MIA, and NAG) displayed greater than additive increases at 24 h following the last exposure as compared to that of the individual metals at the 0.5 mg/m3 exposure levels (Table 3Go). Additivity was no longer apparent by 96 h postexposure, although elevations over control animals persisted. Variability in inflammatory cell counts precluded statistical significance, but the overall trends were consistent with other endpoints (Table 4Go).

Exposure to 1.3 mg/m3 of combined Ni + V led to potentiated delayed bradycardia and hypothermia ({downarrow}160 bpm and {downarrow}4.0°C; Figs. 1C and 2CGoGo) as well as severe increases in arrhythmia frequency (Fig. 3CGo). The duration of the delayed response was similar to that of the response to 1.3 mg/m3 Ni. Additionally, an immediate mild bradycardia ({downarrow}50 bpm) was observed on the first exposure day. The arrhythmia frequency induced at this concentration was over 4 times that induced by the same concentration of Ni alone. The high concentration of Ni + V led to 50% mortality (1 of 4 naïve rats, 3 of 4 telemetried rats); all deaths occurred within 24 h after the final exposure. HR increased acutely prior to death in all lethalities, similar to that reported by Watkinson et al. (2001), and the HR of the remaining telemetried rat was markedly increased (50 bpm above control rats) at the termination of monitoring. The premature lethalities reduced the statistical conclusions that could be drawn from the BALF and hematological endpoints; however, results are included in Tables 3 and 4GoGo.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The results of the present study demonstrate delayed hypothermia, bradycardia, and arrhythmogenesis in rats exposed to aerosolized Ni; concomitant exposure to V exacerbated this effect in a synergistic manner. Previous studies demonstrated that instillation of ROFA in healthy and MCT-treated rats caused both immediate and delayed toxic responses, also consisting of bradycardia, hypothermia, and arrhythmogenesis (Campen et al., 2000aGo; Watkinson et al., 1998Go). When ROFA was inhaled at 15 mg/m3, no immediate response was observed in either MCT-treated or spontaneously hypertensive rats (Kodavanti et al., 1999Go, 2000Go). However, delayed bradycardia and hypothermia were observed in MCT-treated rats beginning after the final exposure to ROFA and lasting 1–2 days (Watkinson et al., 2000Go). Further investigation of these responses revealed that the instilled effects of ROFA were driven primarily by certain transition metals, namely V and Ni. Specifically, instilled V caused the immediate hypothermia, bradycardia, and arrhythmogenesis, while Ni instillation induced similar but delayed effects (Campen et al., manuscript in preparation). It was further demonstrated that Ni potentiated the immediate response to V when instilled concomitantly, but no alteration of the delayed response was apparent. Conversely, in the present inhalation study, the interaction between these metals appears most pronounced in exacerbating the delayed response.

The second highest metals concentration (1.2 mg/m3) in the present study was designed to match the doses used in previous metal instillation studies (Campen et al., manuscript in preparation), and lower concentrations allowed comparisons with other studies. Kodavanti et al. (1999) exposed healthy and MCT rats to 15 mg/m3 ROFA for 6 h/day x 3 days and showed significant increases in BALF LDH and protein levels in healthy rats. The levels of bioavailable Ni and V in 15 mg/m3 ROFA would both be approximately 0.5 mg/m3, according to the composition ratios reported by Dreher et al. (1997). The present study demonstrated further increases in BALF markers of inflammation following exposure to Ni and Ni + V at concentrations comparable to those found in 15 mg/m3 ROFA. Also, no bradycardia or hypothermia was observed in healthy, ROFA-exposed rats, while healthy rats exposed to similar concentrations of just Ni + V in the present study showed significant decreases in both HR and TCO. These differences may be related to the extra day of exposure, to the bioavailability of the metal ions when dissolved and aerosolized as compared to metals that are complexed to PM, or to other attenuating constituents of ROFA. Studies of instilled metals also demonstrated an attenuation by Fe, an additional component of ROFA, on the acute effects of V and Ni + V (Campen et al., manuscript in preparation).

As one of the most important caveats to the instillation method is the immediate and high burden of the bolus dose, it was felt that by prolonging the exposure period to the duration of the delayed response to instilled Ni, the greatest difference between methodologies could be attained. Therefore, the present study shows that the robust immediate response to V previously reported (Campen et al., manuscript in preparation) may be an artifact of the instillation methodology, as only small effects were observed at the highest concentrations of inhaled V and Ni + V after the first day of exposure. However, the delayed effects of inhaled Ni are comparable to those induced by instilled Ni, with regard to the time to onset (2–3 days following initial exposure) and the time to resolution (96 h following the final exposure). The overall duration of Ni-induced hypothermia and bradycardia observed in the present study (96 h) was prolonged compared to that of instillation studies (48–72 h), presumably as a result of the difference in exposure duration.

The "metals hypothesis" proposed by Costa and Dreher (1999) suggests that soluble transition metals may drive the association between increased PM levels and excess daily morbidity and mortality. Several studies have been conducted that demonstrate the in vivo and in vitro toxicity of various metals, including Ni, V, Fe, and Zn (Campen et al., manuscript in preparation; Dreher et al., 1997Go; Dye et al., 1999Go; Kadiiska et al., 1997Go; Kodavanti et al., 1997Go). Such studies primarily highlight the general oxidative effects of the metals as their mechanism of toxicity. Kadiiska et al. (1997) demonstrated that V and Fe have the highest capacity to generate free radicals, as measured by electron spin resonance, while the capacity of Ni was not statistically different from that of saline controls. Dye et al. (1999) reported that ROFA and V, but not Ni, led to altered airway epithelial cell gene expression and cytotoxicity, and furthermore found that the radical scavenger dimethylthiourea could reduce these effects. As these effects were observed at 24 h following initiation of treatment, it is not surprising that effects of Ni were not apparent. Similarly, V (VOSO4 and NaVO3), but not Fe or Ni, induced cytokines in human airway epithelial cells, however, the longest time point examined was 24 h (Carter et al., 1997Go). ROFA and V were shown to elevate protein tyrosine phosphatase levels after a 2-h incubation period in bovine airway epithelial cells, while Ni and Fe had no effects (Samet et al., 1997Go). Epithelial cell lactoferrin receptors were induced 45 min following incubation with Fe and V, but again, not with Ni (Ghio et al., 1999Go). The results of the present study suggest that these in vitro and IT in vivo studies may overemphasize the importance of V and oxidative stress in the toxicity of ROFA, as inhaled V caused minimal effects in healthy rats even at high concentrations. However, since epidemiological studies report strong associations between cardiovascular morbidity and same-day and 1-day lag PM levels, more research is required to identify susceptibility factors that may predispose subjects to the acute effects of V or other PM constituents.

The delayed effects of inhaled NiSO4 seen in the present study are not likely caused by oxidative mechanisms. As mentioned, the oxidative capacity of Ni is much lower than V, and V failed to generate effects on the order of those induced by Ni. There is strong evidence, however, that Ni can interfere with the replicative and repair mechanisms of cells. Dally and Hartwig (1997) demonstrated that while a sufficiently high concentration of Ni could cause oxidative DNA damage, lower doses could inhibit both base and nucleotide excision repair. Ni has also been shown to reduce the action of antioxidants such as catalase, superoxide dismutase, and glutathione (Misra et al., 1990Go; Shainkin-Kestenbaum et al., 1991Go). It is conceivable that at low lung burdens, Ni and/or V could produce oxidative stress that would not pose a significant problem for the defense and repair pathways of a healthy cell. However, the ability of Ni to inhibit antioxidants and repair enzymes could potentiate toxic effects from an oxidant such as V. Moreover, generation of radicals by V could exacerbate the outcome of Ni-induced cell cycle arrest. Either or both of these mechanisms could play a role in causing the synergistic increases in pulmonary inflammation, bradycardia, hypothermia, arrhythmogenesis, and fibrinogen levels seen in the present study.

Bradycardia, hypothermia, and arrhythmogenesis are commonly observed in rodent toxicological studies. A variety of toxic agents, including ozone, chlordimeform, and carbon monoxide (Gautier and Bonora, 1994Go; Gordon et al., 1985Go; Watkinson et al., 1995Go) have been shown to cause these responses in rats and mice. Watkinson and Gordon (1993) defined the hypothermic response as a xenobiotic-induced cluster of effects characterized by hypothermia, bradycardia, and changes in other metabolic parameters, including minute ventilation and physical activity. It was suggested that this response was protective against the toxic effects of most agents, either by reducing the rate of metabolic bioactivation of the compound, or by reducing the uptake of the compound. This response appears specific to rats and other tailed rodents; guinea pigs, for instance, do not appear to display this response (Campen et al., 2000bGo).

It has been proposed that this hypothermic response is parasympathetically mediated, which may explain the increased frequency of certain arrhythmias, including type II atrioventricular node block and premature depolarizations (Watkinson et al., 1998Go). Such a dramatic hypothermia would not likely be induced in humans following exposure to airborne pollutants; however, the initiation of a vagal-response pathway in general may be of great concern to environmental health scientists (Stone and Godleski, 1999Go). There could be early afferent pathways that initiate the rodent hypothermic response that are conserved in humans, though with divergent outcomes. For instance, pulmonary C-fiber activation of central or ganglionic reflexes could plausibly occur in humans following PM exposure, leading to reflex alteration of ventilation and/or cardiac rhythm and conduction. This is particularly relevant given the recent reports of altered heart rate variability associated with PM air pollution in Baltimore, Maryland (Liao et al., 1999Go) and Provo, Utah (Pope et al., 1999Go).

The cardiac arrhythmias observed in the present study, therefore, are quite possibly related to the species-specific hypothermic response. The increased HR in Ni + V-exposed lethalities just prior to death, however, is consistent with heart failure secondary to pulmonary complications. Such lethality patterns were also seen frequently in rats with MCT-induced cardiopulmonary disease when instilled with metals or ROFA (Campen et al., 2000aGo, manuscript in preparation; Watkinson et al., 1998Go, 2001Go). In patients with severe lung disease, secondary disease of the cardiovascular system is a common finding. It is estimated that 20% of COPD patients die from cardiovascular complications, such as heart failure and sudden cardiac death (Fuso et al., 1995Go). The pattern of lethality displayed in this and previous studies suggests that heart failure resulting from respiratory insufficiency is the reason for death in most rats.

Telemetric monitoring has several strengths and weaknesses as compared to traditional toxicological endpoints. The most important strength is the near-continuous nature of data acquisition, which eliminates the need to sacrifice additional animals to obtain measures at specific timepoints. Additionally, the data is acquired in conscious, unrestrained rats, thereby enabling a more natural observation of health effects. One weakness of the telemetric parameters, however, is that markers of inflammation and lung injury from BALF appeared to be more sensitive indicators of metal toxicity. Increases in BALF parameters were observed at 1.7 mg/m3 V and 0.3 mg/m3 Ni, while telemetric endpoints show no change at all in response to V and no response to 0.5 mg/m3 Ni. Nevertheless, the BALF parameters indicate that inhaled V is much less toxic than inhaled Ni, thereby supporting the conclusions drawn from telemetric measurements. Furthermore, the variation in markers of pulmonary injury and inflammation among rats was much higher than that of HR and TCO responses, highlighting the ability of the telemetric approach to obtain significant observations in a reduced number of subjects.

It should be noted that the metal concentrations used in the present study are 2–3 orders of magnitude greater than would be expected even during a high air pollution episode. The daily inhalation dose of nickel on a normal day in a 70-kg human is expected to be only 0.1–1.0 µg (NTP TR 454, 1996), as opposed to the ~ 250 µg doses in 0.3 kg rats from previous IT studies. Occupational studies, however, have measured concentrations of Ni in the range of 0.2–0.8 mg/m3 in Ni refineries (Kiilunen et al., 1997Go), well in the range of concentrations used in the present study. On the other hand, the healthy animals used in the present study may display diminished responses as compared to those anticipated in humans with compromised lung function. The airway deposition fraction of particles in rats is often lower than in humans, due to significant deposition in the turbinates of the rodent nares. Also, in humans, diseased lungs usually have a higher deposition fraction than healthy lungs as a result of slower, yet more turbulent airflow (Kim and Kang, 1997Go). Furthermore, diseased lungs can show deficits in clearance rates, prolonging the exposure time and also increasing the total lung burden. It would not be unreasonable, therefore, to expect adverse responses to much lower metal concentrations in susceptible human subpopulations.

In summary, inhalation of Ni induced delayed hypothermia, bradycardia, and arrhythmogenesis in rats, similar to that observed following Ni instillation. On the other hand, V inhalation produced only mild pulmonary inflammation and minimal physiological responses, much unlike the profound hypothermic responses seen immediately following V instillation. This finding may have important implications for many of the in vitro studies that have demonstrated marked V-induced toxicity. While many factors may serve to amplify the adverse pulmonary effects of V in susceptible individuals, including compromised antioxidant capacity and decreased mucociliary clearance rates, the results of the present whole-body inhalation study argue against an important role for V as an independent toxic constituent of PM. In combination, Ni and V produced synergistic increases in BALF markers of pulmonary inflammation and severely potentiated bradycardia, hypothermia, and arrhythmogenesis. These results help resolve questions regarding the biphasic response to ROFA instillation and suggest pathways by which PM-associated metals may cause the lag effects commonly reported in epidemiological studies. The findings of the present study support the metals hypothesis of PM air pollution adverse health effects, and furthermore indicate that interactions between metals may be key determinants of toxicity.


    ACKNOWLEDGMENTS
 
The authors thank Judy H. Richards for her superb technical assistance on this project. This work was funded in part by U.S. EPA and U.S. EPA/UNC T901915 research training grant.


    NOTES
 
This paper has been reviewed by the National Health and Environmental Effects Research Laboratory of the United States Environmental Protection Agency and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

1 To whom correspondence should be addressed at Division of Pulmonary Medicine, Johns Hopkins Asthma and Allergy Center, Rm. 4a.29, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. Fax: (410) 550-2612. E-mail: mattcampen{at}hotmail.com. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Burnett, L. H., Dales, R., Krewski, D., Vincent, R., Dann, T., and Brook, J. R. (1995). Associations between ambient particulate sulfate and admissions to Ontario hospitals for cardiac and respiratory diseases Am. J. Epidemiol. 142, 15–22.[Abstract]

Campen, M. J., Costa, D. L., and Watkinson, W. P. (2000a). Cardiac and thermoregulatory toxicity of residual oil fly ash in cardiopulmonary-compromised rats. Inhal. Toxicol. 12(S2), 7–22.

Campen, M. J., Norwood, J., McGee, J., Mebane, R., Hatch, G. E., and Watkinson, W. P. (2000b). Ozone-induced hypothermia and bradycardia in rats and guinea pigs exposed in whole-body or nose-only systems. J. Thermal Biol. 25, 81–89.[ISI]

Carter, J. D., Ghio, A. J., Samet, J. M., and Devlin, R. B. (1997). Cytokine production by human airway epithelial cells after exposure to an air pollution particle is metal-dependent. Toxicol. Appl. Pharmacol. 146, 180–188.[ISI][Medline]

Costa, D. L., and Dreher, K. L. (1999). What do we need to know about airborne particles to make effective risk management decisions? A toxicology perspective. Human Ecol. Risk Assess. 5, 481–491.[ISI]

Costa, D. L., and Tepper, J. S. (1988). Approaches to lung function assessment in small mammals. In Toxicology of the Lung (D. E. Gardner, J. D. Crapo, and E. J. Massaro, Eds.), pp. 147–174. Raven Press, New York.

Dally, H., and Hartwig, A. (1997). Induction and repair inhibition of oxidative DNA damage by nickel(II) and cadmium(II) in mammalian cells. Carcinogenesis 18, 1021–1026.[Abstract]

Dreher, K. L., Jaskot, R. L., Lehmann, J. R., Richards, J. H., McKee, J. G., Ghio, A. J., and Costa, D. L. (1997). Soluble transition metals mediate residual oil fly ash induced acute lung injury. J. Toxicol. Environ. Health 50, 285–305.[ISI][Medline]

Driscoll, K. E., Costa, D. L., Hatch, G., Henderson, R., Oberdorster, G., Salem, H., and Schlesinger, R. B. (2000). Intratracheal instillation as an exposure technique for the evaluation of respiratory tract toxicity: Uses and limitations. Toxicol. Sci. 55, 24–35.[Abstract/Free Full Text]

Dye, J. A., Adler, K. B., Richards, J. H., and Dreher, K. L. (1999). Role of soluble metals in oil fly ash-induced airway epithelial injury and cytokine gene expression. Am. J. Physiol. 277, L498–L510.[Medline]

Fuso, L., Incalzi, R. A., Pistelli, R., Muzzolon, R., Valente, S., Pagliari, G., Gliozzi, F., and Ciappi, G. (1995). Predicting mortality of patients hospitalized for acutely exacerbated chronic obstructive pulmonary disease. Am. J. Med. 98, 272–277.[ISI][Medline]

Gardner, S. Y., Lehmann, J. R., and Costa, D. L. (2000). Oil fly ash-induced elevation of plasma fibrinogen levels in rats. Toxicol. Sci. 56, 175–180.[Abstract/Free Full Text]

Gautier, H., and Bonora, M. (1994). Ventilatory and metabolic responses to cold and CO-induced hypoxia in awake rats. Respir. Physiol. 97, 79–91.[ISI][Medline]

Ghio, A. J., Carter, J. D., Dailey, L. A., Devlin, R. B., and Samet, J. M. (1999). Respiratory epithelial cells demonstrate lactoferrin receptors that increase after metal exposure. Am. J. Physiol. 276, L933–940.[Abstract/Free Full Text]

Gordon, C. J., Long, M. D., and Stead, A. G. (1985). Thermoregulation in mice following acute chlordimeform administration. Toxicol. Lett. 28, 9–15.[ISI][Medline]

Kadiiska, M. B., Mason, R. P., Dreher, K. L., Costa, D. L., and Ghio, A. J. (1997). In vivo evidence of free radical formation in the rat lung after exposure to an emission source air pollution particle. Chem. Res. Toxicol. 10, 1104–1108.[ISI][Medline]

Kiilunen, M., Utela, J., Rantanen, T., Norppa, H., Tossavainen, A., Koponen, M., Paakkulainen, H., and Aitio, A. (1997). Exposure to soluble nickel in electrolytic nickel refining. Ann. Occup. Hyg. 41, 167–188.[ISI][Medline]

Killingsworth, C. R., Alessandrini, F., Murthy, G. G. K., Catalano, P. J., Paulauskis, J. D., and Godleski, J. J. (1997). Inflammation, chemokine expression, and death in monocrotaline-treated rats following fuel oil fly ash inhalation. Inhal. Toxicol. 9, 541–565.[ISI]

Kim, C. S., and Kang, T. C. (1997). Comparative measurement of lung deposition of inhaled fine particles in normal subjects and patients with obstructive airway disease. Am. J. Respir. Crit. Care Med. 155, 899–905.[Abstract]

Kodavanti, U. P., Jaskot, R. H., Costa, D. L., and Dreher, K. L. (1997). Pulmonary proinflammatory gene induction following acute exposure to residual oil fly ash: Roles of particle-associated metals. Inhal. Toxicol. 9, 679–701.[ISI]

Kodavanti, U. P., Jackson, M. C., Ledbetter, A. D., Richards, J. R., Gardner, S. Y., Watkinson, W. P., Campen, M. J., and Costa, D. L. (1999). Lung injury from intratracheal and inhalation exposures to residual oil fly ash in a rat model of monocrotaline-induced pulmonary hypertension. J. Toxicol. Environ. Health A 57, 543–563.[ISI][Medline]

Kodavanti, U. K., Schladweiler, M. C., Ledbetter, A. D., Watkinson, W. P., Campen, M. J., Winsett, D. W., Richards, J. R., Crissman, K. M., Hatch, G. E., and Costa, D. L. (2000). The spontaneously hypertensive rat as a model of human cardiovascular disease: Evidence of exacerbated cardiopulmonary injury and oxidative stress from inhaled emission particulate matter. Toxicol. Appl. Pharmacol. 164, 250–263.[ISI][Medline]

Liao, D., Creason, J., Shy, C., Williams, R., Watts, R., and Zweidinger, R. (1999). Daily variation of particulate air pollution and poor cardiac autonomic control in the elderly. Environ. Health Perspect. 107, 521–525.[ISI][Medline]

Misra, M., Rodriguez, R. E., and Kasprzak, K. S. (1990). Nickel induced lipid peroxidation in the rat: Correlation with nickel effect on antioxidant defense systems. Toxicology 64, 1–17.[ISI][Medline]

NTP (1996). Technical Report on the Toxicology and Carcinogenesis Studies of Nickel Sulfate Hexahydrate (CAS No. 10101-97-0) in F344/N Rats and B6C3F1 Mice (Inhalation Studies). Technical Report Series No. 454. NIH Publication No. 96-3370. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, National Toxicology Program, Research Triangle Park, NC.

Ponka, A., and Virtanen, M. (1996). Low-level air pollution and hospital admissions for cardiac and cerebrovascular diseases in Helsinki. Am. J. Public Health 86, 1273–1280.[Abstract]

Pope, C. A., Schwartz, J., and Ronson, M. (1992). Daily mortality and PM10 pollution in Utah Valley. Arch. Environ. Health 42, 211–217.

Pope, C. A., 3rd, Verrier, R. L., Lovett, E. G., Larson, A. C., Raizenne, M. E., Kanner, R. E., Schwartz, J., Villegas, G. M., Gold, D. R., and Dockery, D. W. (1999). Heart rate variability associated with particulate air pollution. Am. Heart J. 138, 890–899.[ISI][Medline]

Rhoads, K., and Sanders, C. L. (1985). Lung clearance, translocation, and acute toxicity of arsenic, beryllium, cadmium, cobalt, lead, selenium, vanadium, and ytterbium oxides following deposition in rat lung. Environ. Res. 36, 359–378.[ISI][Medline]

Samet, J. M., Stonehuerner, J., Reed, W., Devlin, R. B., Dailey, L. A., Kennedy, T. P., Bromberg, P. A., and Ghio, A. J. (1997). Disruption of protein tyrosine phosphate homeostasis in bronchial epithelial cells exposed to oil fly ash. Am. J. Physiol. 272, L426–432.[Abstract/Free Full Text]

Schwartz, J., and Morris, R. (1995). Air pollution and hospital admission for cardiovascular disease in Detroit, Michigan. Am. J. Epidemiol. 142, 23–35.[Abstract]

Shainkin-Kestenbaum, R., Caruso, C., and Berlyne, G. M. (1991). Effect of nickel on oxygen free radical metabolism. Inhibition of superoxide dismutase and enhancement of hydroxydopamine autoxidation. Biol. Trace Elem. Res. 28, 213–221.[ISI][Medline]

Stone, P. H., and Godleski, J. J. (1999). First steps toward understanding the pathophysiologic link between air pollution and cardiac mortality. Am. Heart J. 138, 804–807.[ISI][Medline]

Watkinson, W. P., Campen, M. J., and Costa, D. L. (1998). Cardiac arrhythmia induction after exposure to residual oil fly ash particles in a rodent model of pulmonary hypertension. Toxicol. Sci. 41, 209–216.[Abstract]

Watkinson, W. P., Campen, M. J., Dreher, K. L., Su, W.-Y., Kodavanti, U. K., Highfill, J. W., and Costa, D. L. (2000). Thermoregulatory effects following exposure to particulate matter in healthy and cardiopulmonary-compromised rats. J. Therm. Biol. 25, 131–137.[ISI]

Watkinson, W. P., Campen, M. J., Nolan, J. P., and Costa, D. L. (2001). Cardiovascular and systemic responses to inhaled pollutants in rodents: Effects of ozone and particulate matter. Environ. Health Perspect. 109(Suppl.), 539–546.

Watkinson, W. P., and Gordon, C. J. (1993). Caveats regarding the use of the laboratory rat as a model for acute toxicological studies: Modulation of the toxic response via physiological and behavioral mechanisms. Toxicology 81, 15–31.[ISI][Medline]

Watkinson, W. P., Wiester, M. J., and Highfill, J. W. (1995). Ozone toxicity in the rat: I. Effect of changes in ambient temperature on extrapulmonary physiological parameters. J. Appl. Physiol. 78, 1108–1120.[Abstract/Free Full Text]