Immunotoxicology Branch, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Received February 15, 2002; accepted May 30, 2002
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ABSTRACT |
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Key Words: dioxin; immunotoxicity; host resistance; Reye's Syndrome; influenza infection; pulmonary inflammation.
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INTRODUCTION |
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Humans, particularly young children, may develop Reye's syndrome (RS) when aspirin or certain other therapeutic or xenobiotic chemicals are ingested during influenza or varicella virus infection (Osterloh et al., 1989). Mitochondrial damage and dysfunction (e.g., changes in lipid metabolism and gluconeogenesis, among others) appear to be the underlying cause of the disease (Osterloh et al., 1989
), although increased production of the inflammatory cytokines IL1ß, IL6, and IL8 (reviewed by Visentin et al., 1995
), plus tumor necrosis factor-
(TNF-
), in response to elevated endotoxin levels (due to faulty hepatic endotoxin metabolism) are likely involved in disease pathogenesis (Cooperstock et al., 1975
). Treatment with monoclonal antibodies directed against these mediators has been proposed as a potential treatment for Reye's (Treon and Broitman, 1992
). It is also worth noting that Schecter et al. (1985) reported morphologic alterations in hepatic mitochondria of humans with elevated liver enzymes following accidental exposure to dioxins, PCBs, and furans. In the mouse, nonpermissive infection with large numbers of either influenza A (Sanchez-Lanier et al., 1991
) or B (Schwarz et al., 1991
) virus also produces Reye's-like histologic and metabolic changes in the liver and brain.
Cytokine synthesis patterns in animals exposed to TCDD or infected with influenza, and those detected in human cases of RS are very similar, suggesting that cytokine-induced mitochondrial toxicity and dysfunction may also contribute to mortality in mice exposed to TCDD before infection with influenza. These studies tested the hypothesis that increased mortality rates in TCDD-exposed mice infected with influenza is, at least in part, the result of mitochondrial dysfunction similar to that observed in RS. The diagnostic biochemical criteria for RS include a 1.5 to 3-fold increase in serum ammonia, aspartate aminotransferase (AST), or alanine aminotransferase (ALT), accompanied by hypoglycemia (Crocker and Bagnell, 1981; Osterloh et al., 1989
). Serum ammonia levels increase as intramitochondrial urea cycle enzyme activity decreases; hepatic injury, glycogen depletion, and decreased gluconeogenesis are likewise the result of mitochondrial injury and the subsequent loss of intramitochondrial enzyme activity (Osterloh et al., 1989
). Other biochemical changes often associated with RS include decreased levels of carnitine and changed lipid profiles. Mitochondrial damage decreases ß-oxidation of fatty acids within the organelle, and the resulting shift to cytosolic
-oxidation ultimately leads to depletion of carnitine as it binds to the dicarboxylic acid metabolites produced by this pathway (Engel and Rebouche, 1984
). Biochemical indicators of mitochondrial dysfunction in serum were evaluated, including NH3, AST, ALT, and glucose, as were markers of infection- and TCDD-induced effects on the host.
Based on the work of Kerkvliet and Oughton (1993) and Moos et al. (1994), an alternative hypothesis was also considered, i.e., that the proinflammatory properties of TCDD alone were sufficient to increase mortality in infected mice. Although the mechanism of TCDD-enhanced mortality is unknown, one possibility is that TCDD may act as a proinflammatory agent, and thus contribute to mortality. Deaths from TCDD-induced wasting syndrome (Taylor et al., 1992) or hepatic pathology due to oxidative damage (Alsharif et al., 1994
) can be prevented by injection of antibody to TNF-
, as can the inflammatory response to antigen (sheep erythrocytes) injection in dioxin-exposed animals (Moos et al., 1994
). TNF-
, IL1ß, IL6, and IL8 levels also increase substantially during influenza infection (Van Reeth, 2000
). Thus, TCDD may act additively or synergistically with infection to stimulate the inflammatory response, particularly in the lungs of infected mice. On the other hand, influenza-related mortality was reported following exposure to as little as 0.01 µg TCDD/kg (Burleson et al., 1996
), while the lowest doses of TCDD reported to elevate IL1ß or TNF-
are 1 or 30 µg/kg, respectively, in the rat (Fan et al., 1997
). These findings suggest that while overwhelming inflammation may significantly contribute to mortality at higher dioxin doses, other modes of action may control mortality in mice exposed to lower TCDD doses prior to influenza infection.
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MATERIALS AND METHODS |
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TCDD exposure.
TCDD (purity > 98% by GC-MS, Radian Corp., Austin, TX) was initially dissolved in acetone and then added to corn oil (Sigma Chemical Co., St. Louis, MO). The acetone was removed under vacuum and the stock solution of TCDD diluted with corn oil; dosing solutions were prepared by dilution of stock TCDD in corn oil. Mice were dosed by ip injection of 0.1 ml dosing solution/10 g of body weight, 7 days before infection.
Influenza infection.
A frozen aliquot of influenza A/Hong Kong/8/68 (H3N2) was used to prepare dilutions containing the desired number of plaque forming units (PFU) in 50 µl. Mice were lightly anesthetized with Metofane and infected by intranasal instillation of 25 µl/nostril. Animals were infected with an estimated LD1020 dose of virus.
Experiment endpoints.
In all experiments, mice were weighed, sacrificed by pentobarbital overdose, and lung weights were determined.
Blood was collected from the heart, and serum samples were frozen for later analysis of biochemical indicators of mitochondrial damage. These included ammonia, glucose, carnitine, cholesterol, triglycerides, ALT, and AST. Analysis was done using a Cobas Fara II centrifugal spectrophotometer (Hoffman-La Roche, Branchburg, NJ).
When virus titers, pulmonary leukocytes, and soluble constituents were to be analyzed, lungs were lavaged with warmed (37°C) sterile Hanks Balanced Salt Solution. Cells were isolated by centrifugation, counted, and cytocentrifuge (Cytospin Model II, Shandon, Pittsburgh, PA) slides were prepared and stained for differential cell counts. The lavage supernatant was frozen for later analysis of total protein and lactate dehydrogenase (Cobas Fara II analyzer), cytokines (ELISA kits from R&D Systems, Minneapolis, MN), and determination of virus titer. In cases where the ELISA assay detected no cytokine protein, data were presented as the limit of detection value as the group mean, to allow statistical analysis of data from all groups. The lungs were then aseptically removed, weighed, and frozen for later determination of virus titer. To estimate pulmonary virus load, lung tissue was ground with sterile sand, using a mortar and pestle, and dilutions of lung homogenate were plated on confluent cultures of Madin Darby canine kidney (MDCK) cells. Aliquots of BALF were likewise diluted and plated on MDCK cell monolayers. After 48 h of incubation, the monolayers were stained with crystal violet; plaques were counted and virus titers calculated. Results are expressed as PFU/lung, i.e., as the sum of PFU in lung tissue and in BALF.
Experimental design.
Preliminary experiments were conducted to determine whether either TCDD exposure alone or viral infection alone affected the serum clinical chemistry values known to be altered in cases of RS. Groups of animals were dosed with TCDD (0.0011.0 µg/kg) or infected with influenza virus (25, 50, 100, or 200 PFU) and killed on days corresponding to days 1, 7, and 10 of infection (i.e., 8, 14, and 17 days after TCDD dosing was done or would have been done).
The effects of TCDD exposure on infection-induced mortality were evaluated at doses (0.0011.0 µg/kg) previously reported to increase susceptibility to influenza (Burleson et al., 1996; House et al., 1990
) when given 7 days before infection. Groups of 3 control and 3 TCDD-exposed mice were killed 1, 7, and 10 days after infection for evaluation of the endpoints described below. Separate groups of 6 animals from each dose group were observed for mortality.
Mortality rates were not affected by doses 1 µg TCDD/kg; thus, the effects of a broader range and higher dose (0, 0.025, 0.5, or 10.0 µg/kg) of TCDD exposures on mortality were determined in dedicated groups of 20 mice. Additional groups of mice were also exposed to TCDD and killed at the estimated midpoint of infection to evaluate serum, BALF, and lung tissue for indications of mitochondrial damage or enhanced lung inflammation. Subgroups of mice injected with corn oil or the highest dose of TCDD, but not infected with influenza, were included in this experiment as additional controls and killed 11 days after chemical injection (equivalent to day 4 of infection) to monitor the effects of TCDD alone.
Statistical analysis.
Significant differences in the proportion of mice recovering from influenza infection was determined by Chi-squared analysis, and two-way ANOVA was used to determine whether the dose of TCDD was related to survival time. Other data were tested using two-way ANOVA when appropriate; significant effects were evaluated using Tukey's test to determine which groups differed from each other.
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RESULTS |
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As shown in Figure 3, exposure to 10 µg TCDD/kg significantly decreased the percentage of mice recovering from infection, but did not shorten the interval between infection and onset of mortality. There was no difference in mortality between groups given lower doses of TCDD and all groups of infected mice had similar virus titers, ranging from 5.8 to 5.9 log10 PFU/lung. Body weight was similar in all groups (data not shown); as before, lung weights were increased by infection, but not by exposure to TCDD (not shown).
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DISCUSSION |
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The biochemical diagnostic criteria for RS include a 1.5 to 3-fold increase in serum ammonia, AST, or ALT, accompanied by hypoglycemia (Crocker and Bagnell, 1981; Osterloh et al., 1989
). Elevated NH3 levels have also been reported in a mouse model of RS (Crocker et al., 1986
). However, we found that serum ammonia levels were not elevated by TCDD exposure alone (Table 1
), virus infection alone (Table 2
), or combined exposure and infection (Table 3
). Serum transaminase levels were elevated in TCDD-exposed mice infected with influenza, but were similarly elevated by either infection or TCDD exposure alone, suggesting no interaction between chemical exposure and influenza infection. Hypoglycemia was observed 7 days after infection of mice with
50 PFU, but not in groups only exposed to TCDD or in combined TCDD exposed/infected (25 PFU) mice. Other biochemical changes often associated with RS include decreased levels of carnitine and changed lipid profiles (Engel and Rebouche, 1984
). In these studies, decreased carnitine levels were observed in mice dosed with 10 µg TCDD/kg alone, but not in animals given the same dose and infected 7 days later, contrary to what would be expected from an interaction between chemical exposure and viral infection. We also found that a large challenge dose (200 PFU) of influenza alone increased cholesterol levels 7 days after infection, whereas exposure to 1 µg TCDD/kg 7 days prior to infection with 25 PFU decreased cholesterol levels 1 day after infection. Triglyceride levels were similar in experimental and control mice in all experiments. When considered together, these results provide strong evidence that RS-like mitochondrial dysfunction did not occur, even at the highest dose of TCDD; thus, the hypothesis that a Reye's-like syndrome is the cause of increased mortality must be rejected.
An alternative hypothesis was also considered, i.e., that TCDD was acting as a proinflammatory agent. Others have reported increased influenza mortality without an increase in virus titers in mice exposed to other xenobiotics. For example, Selgrade et al. (1988) reported similar viral titers in controls and ozone-exposed mice, although mortality was twice as high and survival time was decreased in O3 versus air controls. Lung weight was also increased and pulmonary function was decreased in exposed, infected mice; the authors therefore suggested a possible synergistic interaction between ozone exposure and infection that resulted in greater morbidity and mortality. Ryan et al. (2000) also reported that exposure to ultraviolet B (UVB) light had similar effects on the susceptibility of mice, i.e., increased mortality in the absence of increased viral titers.
Although dioxin and ozone are quite different chemically and toxicologically, it is plausible that these xenobiotics share a mode of action underlying increased sensitivity to viral infection. Influenza infection alone produces significant pulmonary inflammation, characterized by an influx of PMNs (Sweet and Smith, 1980), and elevated levels of proinflammatory cytokines including TNF-
, and interleukins-1 (
and ß), -6, and -8 (van Reeth, 2000
). Ozone exposure likewise stimulates pulmonary levels of inflammatory cytokines, particularly TNF-
, IL1ß, and IL6 (reviewed by Ryan, 2000
). TCDD exposure has been reported to elevate hepatic levels of TNF-
and IL1ß as well (Fan et al., 1997
), although others have reported that circulating levels of inflammatory cytokines are unaffected by TCDD (for example, Nishimura et al., 2001
). In our experiments, TCDD exposure alone did not elevate levels of proinflammatory cytokines, but TNF-
, MIP-1
, and MIP-2 levels were elevated by preinfection exposure to TCDD, consistent with a greater pulmonary inflammatory response to infection in exposed mice. It is not clear why TNF-
and MIP-1
were only increased by the 0.5 µg TCDD/kg dose. PMN influx was also significantly increased in exposed, infected mice, a finding previously reported by Warren et al. (2001). The common thread for these disparate xenobiotics appears to be an enhanced inflammatory response that may, in combination with the inflammation induced by influenza infection, overwhelm the host. Support for this concept is provided by studies reporting that immunosuppression may increase survival rates following influenza infection. For example, both restraint stress (Hermann et al., 1993
) and in utero exposure to chlordane (Menna et al., 1985
), increase survival of adult mice challenged with influenza. These results suggest that immunosuppressive agents lacking an inflammatory component increase survival by down-regulating pulmonary inflammation, and lend support to the hypothesis that TCDD increased influenza mortality by increasing pulmonary inflammation.
In these experiments, survival of infected mice was decreased by exposure to 10 µg TCDD/kg 7 days prior to infection with influenza A virus, although virus titers were not increased. Our results are similar to those of Burleson et al. (1996) in that mortality was not accompanied by increased virus titers, and reinforce the observation that unrestricted virus replication secondary to TCDD-mediated immunosuppression is not responsible for mortality. Clinical chemistry endpoints considered critical to the diagnosis of RS did not support our hypothesis that RS-like mitochondrial dysfunction plays a role in mortality. Instead, our results suggest that the proinflammatory action of TCDD has a more profound adverse effect on survival than does dioxin's potent immunosuppressive action in mice.
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ACKNOWLEDGMENTS |
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NOTES |
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This report has been reviewed by the U.S. Environmental Protection Agency's Office of Research and Development, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
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