The findings reported by Andreazza et al. (2007)
of elevated levels of DNA damage, measured by the Comet assay, in cells of the blood and brain of adult rats exposed to MPH for 28 days were not confirmed in our study. Our findings are consistent with the accumulating evidence that MPH does not induce genetic damage in vitro
or in vivo
(Mortelmans et al., 1986
; Teo et al., 2003
; Suter et al., 2006
, Walitza et al., 2007
; Manganatha et al., 2008; Witt et al., 2008b
; Walitza et al., 2009
; Morris et al., 2009
A number of factors may be responsible for the differences in results between our study and that reported by Andreazza et al. (2007)
. One difference is the method of scoring DNA damage (Comet figures). Andreazza et al. (2007)
visually evaluated 100 randomly selected cells and subjectively scored the images according to tail intensity (determined by the size and shape of the tail). In contrast, we used an automated system that sequentially evaluated well-defined Comet figures using objective quantification of image pixels that were converted into measurements of tail length and % tail DNA; we then quantified DNA damage using the OTM (Olive Tail Moment) measure, which factors in both tail length and % tail DNA (Burlinson et al., 2007
). In addition, cells containing LMW DNA () were evaluated separately in our study to assess any possible impact of cytotoxicity on interpretation of the Comet data. It should be noted that we used flash freezing of tissue samples in DMSO to carefully control the time interval from obtaining tissue until lysing. The low frequency of cells with LMW or fragmented DNA (e.g.
<2% in the liver of vehicle control mice and no apparent increase in MPH exposed mice) demonstrates that our procedure for flash freezing produces little nuclear DNA degradation. This observation is consistent with an earlier report of a direct comparison of fresh and cryopreserved lymphocyte samples in which there was no difference in the amount of DNA damage detected by the Comet assay (Duthie et al., 2002
). Moreover, there is no expectation that the extent of DNA damage induced by snap freezing will vary among samples that are processed similarly. Thus, our data benefit from the careful control of the time between tissue harvest and lysis, a critical factor for obtaining consistent, reliable data using the Comet assay.
The study protocol we employed differed in certain other key respects from the one used by Andreazza et al. (2007)
. Rather than administering MPH by intraperitoneal injection, which avoids first-pass metabolism, we employed oral gavage, a route that better mimics the route of exposure (oral) and metabolism of MPH in humans. Recent publications provide evidence that metabolism of MPH may be strongly influenced by route and treatment regimen (e.g., oral versus dermal patch, and extended release formulations versus single daily bolus dosing), and therefore, using a route (gavage) that more closely approximates human oral dosing might be expected to yield results more reflective of events that occur in humans (Teicher et al., 2006
; Devilbliss and Berridge, 2008).
In regards to dose selection, we used the two highest doses administered in the Andreazza study (2 and 10 mg/kg/day), and we added a third, higher dose (25 mg/kg/day). This higher dose was used to compensate for any reduced bioavailability of MPH administered via the gavage route (our study) compared with IP injection (Andreazza et al. study), and enhance our ability to detect DNA or chromosomal damage, were either to occur as a consequence of MPH exposure. With regards to bioavailability, Gerasimov et al. (2000)
measured concentrations of MPH (a racemic mixture of d
- and l
-MP) in plasma, and in striatum and cerebellum of Sprague-Dawley rats 20 minutes after either intraperitoneal or intragastric administration of a single dose of 5 mg/kg MPH and found higher concentrations of MPH (approximately 2.5-fold) in all three tissues following intraperitoneal dosing compared with the oral route. Studies by Wargin et al. (1983)
comparing bioavailability of MPH in rats and monkeys following a single intravenous or oral gavage administration indicated that both routes had similar bioavailability values of approximately 20% in both species, based on area-under-the-curve measurements in plasma over an 8 hour period of time (Wargin et al., 1983
). Based on this limited information, use of the gavage route in our study was unlikely to have appreciably decreased the total exposure of the rats in our study to MPH, although the kinetics of exposure may have been markedly different than the kinetics in the Andreazza et al. (2007)
study. Our use of the higher 25 mg/kg dose would be expected to compensate to some degree for a decrease in MPH bioavailability from oral administration.
In another point of contrast, we used only adult male Wistar Han rats rather than adults and juveniles, because increases in DNA damage reported in the earlier study were stronger and more consistent in the adult animals. Furthermore, we used only the 28-day repeated exposure protocol, because Andreazza et al. (2007)
reported more pronounced increases in DNA damage in 28-day exposed rats compared with rats that received a single injection of MPH. Another justification for using the repeated dosing protocol is the closer similarity to human exposures to MPH, which are typically chronic rather than single administration.
Because Andreazza et al. (2007)
reported DNA damage in the hippocampus and striatum of the brain following 28 days of exposure to MPH, we added a histology component to our protocol to look for pathological changes associated with MPH exposure in tissues from these brain regions, where MPH is believed to exert its clinical effects in humans (Biederman and Faraone, 2005
; Dommett et al., 2008
). No evidence of histopathological changes were seen in either region in any of the dose groups in our study, and likewise, no histopathological changes were noted in tissues of the frontal cortex, another brain region believed to be a site of MPH therapeutic activity (Biederman and Faraone, 2005
; Devilbiss and Berridge, 2008
In addition to the absence of primary DNA damage in our study, no significant increases in MN-RET (micronucleated reticulocytes) were seen in MPH-treated rats, a result that is consistent with the negative results reported by Andreazza et al. (2007)
and others for induction of micronuclei either in lymphocytes or in erythrocytes of MPH-treated rats, mice, or nonhuman primates (NTP, 1995
; Suter et al., 2006
; Teo et al., 2003
; Manjanatha et al., 2008
; Morris et al., 2009
). Assessment of MN frequencies in rat RET is a relatively new procedure (Torous et al., 2000
), and was facilitated by the advent of flow cytometry for evaluating micronucleated erythrocyte frequencies in peripheral blood (MacGregor et al., 2006
). Thus, although the rat spleen rapidly and efficiently removes micronucleated erythrocytes from circulation, evaluating damage in very young RETs, newly emerged from the bone marrow compartment, provides an accurate assessment of recently acquired chromosomal damage (Witt et al., 2008a
). Using flow cytometry, RETs are identified by the presence of an active transferrin receptor (CD71+) on the cell surface; newly emerged RETs have the highest CD71 expression levels, and mature erythrocytes are CD71−. The negative micronucleus test results in MPH-treated rats in the study reported here support the results of studies conducted in humans in which no increase in the frequency of micronucleated lymphocytes was observed following exposure to therapeutic doses of MPH for periods of time ranging from 1 month to 2 years (Walitza et al., 2007
; Walitza et al., 2009
; Witt et al., 2008b
; Tucker et al., in press
). Although the numerous MN studies conducted on humans and animals treated with MPH have used varied exposure times and experimental protocols, and have assessed damage in different cell types (reticulocytes, erythrocytes, or lymphocytes), the consistency of the negative responses across all studies strongly suggests that MPH does not induce numerical or structural chromosomal damage in vivo
Rarely, adverse cardiovascular events (tachycardia, sudden death) have been reported in children taking MPH and other stimulant medications for treatment of ADHD (Beiderman et al., 2006). Therefore, in addition to the genetic toxicity and brain histopathology studies, we examined biomarkers of cardiac injury including serum levels of cardiac Troponin T, Troponin I, and Fatty Acid Binding Protein-3 in rats exposed to MPH. Cardiac troponins are sensitive biomarkers of cardiac injury, and have recently gained increasing importance as part of an overall assessment of cardiac toxicity (Gaze and Collinson, 2005
). No evidence of cardiac injury was observed in any of the MPH-treated rats using these endpoints of damage, and histological examination of the hearts revealed no treatment-related effects.
The negative results reported here from the MN and Comet assays add to the rapidly growing body of evidence that MPH, at clinically relevant and higher doses, after acute or prolonged exposure periods, in a variety of in vivo test systems, does not induce cytogenetic damage. Together, results of all these recently conducted studies should alleviate concerns regarding the potential for genetic damage from MPH and other stimulant drugs used in the treatment of ADHD. Answers to questions regarding the possibility of changes in normal patterns of behavior, growth and maturation, or learning associated with chronic exposure to MPH during development await further investigation.