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Department of Human Biology and Nutritional Sciences and
*
Department of Pathobiology, University of Guelph, Guelph, ON, Canada N1G 2W1;
Laboratory for Skin Cancer Research, Hospital Research Center of University Laval, Ste. Foy, QC, Canada G1V 4G2; and
**
Unit of Health and Environment, Hospital Research Center of University Laval, Ste. Foy, QC, Canada G1V 4G2
2To whom correspondence should be addressed. E-mail: jkirklan{at}uoguelph.ca.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • niacin deficiency • leukemia • nitrosourea • poly(ADP-ribose) • chemotherapy • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–3
). The increase in risk of developing a secondary leukemia is of the order of 10- to 100-fold over the general population and 3- to 10-fold over patients treated by radiotherapy (2
).
Nitrosoureas were used more extensively in the past but are still useful in the treatment of certain tumors (4
). Nitrosoureas spontaneously decompose to form two reactive species, an alkylating group and a carbamoylating group, both of which may react with DNA (4
), RNA and protein. Unfortunately, nitrosoureas cause marked and often prolonged bone marrow suppression (4
) and are one of the chemotherapy drugs most strongly associated with the induction of secondary cancers (3
). Ethylnitrosourea (ENU)3
is a monofunctional ethylating agent that we have used as a simple model of the more complex chemotherapeutic nitrosoureas. The monofunctional nitrosoureas are potent leukemogens that model the leukemogenic aspect of chemotherapy agents quite well, especially when used in certain strains of rats such as Long-Evans, which respond with primarily nonlymphoblastic leukemias (5
). The alkylating capacity of nitrosoureas is primarily responsible for their leukemogenic potential, due to the low content of alkyltransferase protein in the bone marrow (6
). Alkylation of DNA occurs relatively homogeneously throughout the body due to the nonenzymatic formation of the reaction product. This is similar to most chemotherapy drugs, which generally cause DNA damage without cytochrome P450-mediated bioactivation.
If a methyl or ethyl adduct is not removed by alkylguanine transferase, the whole base adduct may be removed via excision repair. Removal of the modified base and neighboring nucleotides creates a strand break, which activates the nuclear enzyme, poly(ADP-ribose) polymerase (PARP). Active PARP utilizes NAD+ as a substrate in the synthesis of poly(ADP-ribose). PARP is usually the major acceptor protein, but a variety of other nuclear proteins become modified to some extent (7
). The cloud of negatively charged poly(ADP-ribose) at the site of DNA damage may play several roles, including regulation of excision repair, p53 function and apoptosis (8
). The most consistent evidence, however, shows that poly(ADP-ribose) synthesis prevents recombination events at the site of damage, probably by repelling other DNA strands, thereby decreasing the risk of chromosomal translocations (8
,9
). This is critical in the bone marrow because most leukemias are caused by translocation events of this nature (10
).
NAD+ synthesis is dependent on dietary niacin, and deficiency in this vitamin has been shown to dramatically inhibit DNA repair in cell culture models (11
,12
). However, there is limited knowledge of its effects in the whole animal during exposure to DNA damaging agents. Niacin is a nutrient that appears to be deficient in many chemotherapy patients (13
), and chemotherapy can induce pellagra (clinical niacin deficiency) in cancer patients (14
). It is important to clarify the effects of niacin status on chemically induced leukemogenesis as a first step toward recommendations regarding niacin status and the chemotherapy patient.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Ethylnitrosourea, Modified Wright Giemsa Stain and reagents for NAD analysis were purchased from Sigma Chemical (St. Louis, MO). Halothane, B.P. anesthetic was obtained from MTC Pharmaceuticals (Cambridge, Canada). Isotone II and Zap-oglobin II were purchased from Coulter Electronics of Canada, (Burlington, Canada). Buffered formalin (100 g/L) was purchased from Fisher Scientific (Ottawa, Canada). Reagents for gel electrophoresis were obtained from BioRad Laboratories (Mississauga, Canada).
Animals.
In previous studies, treatment of Long-Evans rats with propylnitrosourea caused a spectrum of leukemias very similar to those induced in humans by alkylation-based chemotherapy (5
). Due to the unavailability of propylnitrosourea, we designed a model based on ENU treatment of Long-Evans rats. Animals were housed and treated in accordance with the guidelines of the Canadian Council on Animal Care. Male Long-Evans rats were obtained at weaning (40–50 g) (Charles River Canada, St. Constant, Canada), housed in suspended wire-bottomed cages and given free access to water with a 12-h light:dark cycle. Feed intake was determined daily.
Diets.
Rats (21 d old) were fed a niacin-deficient (ND) purified diet or pair-fed (PF) identical quantities of the same diet supplemented with exogenous nicotinamide at 0.03 g/kg diet (normal requirement of rats for niacin). The diet, referred to as 7/6, is based on a mixture of 7% casein and 6% gelatin (15
). The mixture of gelatin with casein is required to limit the content of tryptophan, which may be used in an alternate pathway for NAD synthesis. As the rats become niacin deficient, they consume less food. To prevent differences in intake of nutrients other than niacin, the control rats are pair-fed (PF), i.e., they were placed in groups of similar weight, with one rat in each of 4 treatment groups, and all were fed the quantity of food consumed by the ND rat the previous day. All treatment groups maintained positive growth rates.
Chemotherapy protocol.
Using ENU, we simulated a chemotherapy protocol in nontumor-bearing rats. Rats were gavaged with 12 doses of either 30 mg ENU/kg body in water (pH 4.0) or an equivalent volume of water (pH 4.0) alone (CON). Beginning at 28 d of age, each rat was gavaged every 2 d until 50 d of age. As mentioned above, all rats were pair-fed to the ND-CON, which generally had the lowest voluntary food intake. After the last ENU treatment, all rats were then fed the AIN-93M diet (16
) and monitored for the development of tumors or for weight loss. This model approximates the treatment of a cancer patient chemotherapy followed by a recovery period with a return to a higher quality diet. Rats were fed an equal amount of diet each day, based on body weight, to ensure similar energy intakes throughout the remainder of the trial.
Cancer development.
Rats that developed palpable tumors > 1 cm in diameter or lost >5% of their maximal body weight were killed, and necropsies were performed to determine the nature of the cancer(s) involved. Although this is not technically a mortality design, it does allow a quantifiable end point with a minimum of animal suffering. We will refer to these results as morbidity curves. The rats were gaining weight normally throughout the experiment, and a 5% loss in body weight was almost invariably associated with an advanced neoplastic state of some kind. A large majority of rats were terminated on the basis of the weight loss criterion, with only a few developing palpable tumors > 1 cm in the absence of 5% body weight loss. Tissue sections were fixed in buffered formalin (100 g/L) and then embedded in paraffin, sectioned, affixed to slides and stained with hematoxylin and eosin. Control rats were killed at 52 wk of age and autopsies were performed.
Acute response to ENU; NAD+, poly(ADP-ribose) and DNA strand break analysis.
Initially, we conducted a time course in control (niacin-replete) rats, with bone marrow samples taken at 1.5, 3, 6, 12 and 24 h after ENU treatment. On the basis of an early peak in poly(ADP-ribose) levels at 3 h, this time point was used to determine the effect of niacin deficiency. After 3 wk of consumption of niacin-deficient or pair-fed niacin adequate diets, rats were treated with a single dose of ENU or vehicle, as described earlier in this paper, except that the dose used was 100 mg/kg body rather than 30 [to create larger amounts of poly(ADP-ribose) for ease of quantification]. Bone marrow was processed as follows. Both femurs were dissected free and severed at the condyle; each marrow was flushed with 4 mL ice-cold PBS containing protease inhibitors (10 mg/L leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride, 17.5 mg/L aprotinin). A 25-µL aliquot from each sample was saved for cell counting, before and after lysing of RBC.
For analysis of NAD+, the cell suspension from one femur was acidified with concentrated perchloric acid to obtain a final concentration of 1 mol/L. Precipitated proteins were removed by centrifugation at 12,000 x g for 10 min, and the acidic supernatant was frozen. On removal, samples were neutralized with 2 mol/L KOH, and analyzed for NAD+ content via the enzyme cycling of alcohol dehydrogenase (17
).
For analysis of poly(ADP-ribose), cells from the second femur were centrifuged in a refrigerated microfuge at 12,000 x g for 1.5 min, resuspended by vortex in 50 µL PBS, lysed in 500 µL of loading buffer (6 mol/L urea, 100 g/L glycerol, 10 mmol/L Tris base, 20 g/L SDS, 0.2 g/L bromphenol blue, 50 g/L ß-mercaptoethanol) and frozen in liquid nitrogen. This protocol was designed to obtain an inactivated cell extract as quickly as possible, given that poly(ADP-ribose) has a short half-life in whole cells. After thawing, samples were sonicated for 45 s, separated by 8% SDS-PAGE and transferred to nitrocellulose (Hybond-C pure, Amersham, Baie d’Urfé, Canada). The membrane was blocked for 1 h in PBSMT (PBS, 50 g/L dried milk powder, 10 g/L Tween), incubated with 96–10 antibody (1/5000) in PBSMT overnight at 4°C and for 2 h with anti-rabbit peroxidase-linked antibody (1/30,000) (Cedar Lane Laboratories, Hornby, Canada). The 96–10 antibody is a polyclonal prepared in rabbits against bovine serum albumin (BSA)-linked poly(ADP-ribose) (18
). After washing, treatment with ECL+ (Mandel Scientific, Guelph, Canada) and film exposure, the smears of poly(ADP-ribose) were quantified using thresholding functions of Northern Exposure densitometry software. This type of gel shows total poly(ADP-ribose) content (total lane density), the MW of protein acceptors (lower edge of bands) and gives some idea of poly(ADP-ribose) chain length or numbers per protein (the length of the smears above proteins) (19
).
DNA strand breaks were detected using the fluorometric method of Birnboim and Jevcak (20
). Briefly, bone marrow cell lysates (25 x 109 cells/L) were exposed to alkaline-induced DNA unwinding conditions (0.2 mol/L NaOH to give pH 
12.8). In the presence of DNA strand breaks, unwinding occurs more rapidly, leaving less double-stranded DNA intact. The degree of unwinding in a set period of time (60 min) was monitored using the fluorescent dye, ethidium bromide (Aldrich Chemical, Milwaukee, WI), which binds selectively to double-stranded nucleic acids. Total DNA was measured in a set of samples (T tubes) using a neutralizing solution first to protect the DNA from unwinding. The level of strand breaks in vivo was estimated from the disappearance of fluorescence in the alkali-treated sample, indicating the amount of single-stranded DNA (ssDNA) generated in vitro due to unwinding from points of DNA strand breakage. Background fluorescence (e.g., due to duplex RNA) was corrected for by measuring the fluorescence in a set of samples sonicated first to ensure rapid and complete denaturation of DNA in alkali. Fluorescence was measured using a Hitachi F-2000 spectrofluorometer (excitation 520 nm, emission 590 nm; Toronto, ON).
Analysis of data.
Morbidity curves were analyzed by probit and 
2 analyses. Differences in growth, NAD+ and poly(ADP-ribose) among treatment groups were analyzed by two-way ANOVA using the General Linear Modeling procedure (SAS/PC, SAS Institute, Cary, NC) and Duncan’s New Multiple Range test. Differences in benign lesions were analyzed by Fisher’s Exact test, whereas malignancies were analyzed by t test (because some rats had more than one malignancy). In all cases, differences with P < 0.05 were considered significant. ANOVA and Duncan’s analyses were not applied to the data in Figure 5
due to differences in variability associated with treatment means.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Reduced intake did occur and rats were pair-fed accordingly, preventing any differences in body weight during the ENU and diet treatment period. From 5 to 8 wk of age, ND rats exhibited classical symptoms of pellagra as described previously in this laboratory, including alopecia, dermatitis, diarrhea and ataxia (15
). Interestingly, ND-ENU rats exhibited dramatically less severe external signs of niacin deficiency than the ND-CON rats (visual observation). When they consumed the AIN-93M diet, the growth of rats was more rapid than during the first 4 wk of the experiment and did not differ among groups, reflecting the use of body weight to allocate daily quantities of food and adequate recovery of rats from niacin deficiency and ENU treatment.
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) The latency time to 50% morbidity (LT50) was 5.7 wk shorter in ND rats (LT50 for PF = 30.6 wk, LT50 for ND = 24.9 wk, 2 = 63.1). The difference between treatments was greatest in the early stages, with a difference of 10 wk between the morbidity curves at a cancer incidence of 20% (LT20)(Fig. 2)
. These differences occurred during a total interval of only 23 wk, encompassing 0–100% mortality.
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Table 1
lists the pathologies that were found in rats at the end of the morbidity study. All of the ND rats had at least one malignancy, whereas the PF rats displayed a mixture of malignant and benign neoplasms, and one case of unidentified etiology. It was clear that the morbidity in both groups was due almost completely to neoplastic conditions. Leukemias and lymphoproliferative disorders formed the majority of cancers in this model, and played a major role in the early differences in morbidity between ND and PF rats. In the first 20 wk after ENU treatment, ND rats developed 17 malignancies, including 11 leukemias, whereas PF rats developed 3 malignancies including 2 leukemias.
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. Total benign growths (epithelial + stromal) were more frequent in the PF than the ND rats (two-tailed Fisher’s Exact test). Niacin deficiency also caused a 47% increase in the average number of malignancies per rat (1.316 ± 0.11 and 0.895 ± 0.15 malignancies/rat in ND and PF groups, respectively, two-tailed t test). This was in spite of the fact that ND rats had a shorter time in which to develop tumors due to the more rapid onset of morbidity (Fig. 2)
. Paralysis was associated with morbidity in three rats of each treatment group. Spinal cord analysis showed that two of three cases of paralysis in the ND-ENU group were associated with spindle cell tumors in the nervous system. Some other cases of paralysis were associated with granulocytic leukemia and a marked leukemic infiltration of the spinal cord.
Niacin deficiency caused dramatic changes in blood and bone marrow NAD+ concentrations (Fig. 3
). Niacin deficiency decreased blood NAD+ by 65% (Fig. 3
A). Blood NAD+ was not altered by ENU treatment (data not shown), which was expected due to the lack of DNA and PARP in red blood cells, which carry the majority of blood NAD+. Bone marrow NAD+ decreased by 80% in niacin-deficient rats (Fig. 3
B). There was no significant effect of ENU treatment on bone marrow NAD+ (data not shown), which may have occurred if DNA damage and poly(ADP-ribose) synthesis were taking place in the bone marrow cells.
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shows an immunoblot and densitometry time course of bone marrow poly(ADP-ribose) levels following a single dose of ENU (100 mg/kg body). This assay uses a polyclonal antibody (96–10) raised to a poly(ADP-ribose)-BSA conjugate, as described earlier (18
). There is a peak in poly(ADP-ribose) accumulation at 3 h, followed by a rapid drop through 6 and 12 h and a small, but reproducible increase at 24 h. We selected 3 h as the time point for comparison of the different dietary groups.
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shows immunoblot analysis of protein-bound poly(ADP-ribose) in PF and ND rats, with and without ENU treatment. For purposes of quantification, standards of control and diethylnitrosamine (DEN)-treated liver were created in vivo as previously described (21
) and quantified by HPLC analysis [control and DEN-treated liver samples contained 45 and 227 fmol of poly(ADP-ribose) per lane]. This technique provides information on the total quantity of poly(ADP-ribose) (total density of lane), molecular weight of major acceptor proteins (lower edge of distinct bands) and, to some extent, the length of poly(ADP-ribose) chains (the length of the smear above a clear acceptor protein) (19
). For example, the large smear of reactivity originating at 116 kDa in the DEN-treated liver sample represents the automodification of poly(ADP-ribose) polymerase by multiple long polymers (18
). The blot in Figure 5
used composite samples from four different rats in the same treatment group. Those samples were also run individually on separate blots; after background subtraction and thresholding, the total gray in each lane was determined. The known values for the liver samples from HPLC analysis were applied as standards to the bone marrow samples. The bone marrow lanes were loaded by nucleated cell number and they appeared identical (loading density and pattern of bands) by fast green staining before immunoblotting. The densitometry analysis was used to prepare the graph in Figure 5
.
Niacin deficiency alone (ND-CON) caused a dramatic decrease in basal poly(ADP-ribose) levels in the bone marrow, to the point that the smears were not detectable by densitometry (Fig. 5)
. ENU treatment alone (PF-ENU) caused a fivefold increase in poly(ADP-ribose) levels in PF rats (Fig. 5)
. There was a small induction by ENU of poly(ADP-ribose) formation in the niacin-deficient bone marrow (ND-ENU), but it was still below the level of the controls before ENU treatment. Basal poly(ADP-ribose) was concentrated in smears that originated at 116 and 90 kDa, and likely represented automodification of PARP and its proteolytic fragment (commonly associated with apoptosis) (22
). There was some reactivity at 66 kDa, which may represent albumin cross-reactivity, due to the use of a poly(ADP-ribose)-BSA conjugate in raising the antibody. ENU treatment caused widespread formation of poly(ADP-ribose), with strong labeling in the 90- and 116-kDa regions, and diffuse labeling through to very-low-molecular-weight acceptors. There was a distinct band between 116 and 205 kDa, which was often obscured by the 116-kDa smear. The liver had a much clearer acceptor pattern for basal poly(ADP-ribose). DEN-induced synthesis occurs almost exclusively in the 116-kDa smear. Interestingly, the band corresponding to 66 kDa also responded to DEN treatment in the liver, suggesting that it may be a protein modified by poly(ADP-ribose). If this band does represent cross-reactivity with rat albumin, it could reflect changes in albumin synthesis or accumulation in response to DEN.
There was an identical increase in alkaline-released ssDNA 3 h after ENU treatment in PF and ND rats (Fig. 6
). The 3-h time point was chosen because it was the peak in poly(ADP-ribose) accumulation. At 24 h, the amount of alkaline-released ssDNA in PF rats had returned to basal levels and was significantly lower than the 3-h time point. In the ND rats, the ssDNA at 24 h was not significantly greater than basal, but also not significantly less than the 3-h time point.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,8
,11
,12
,23
). Such studies have provided valuable information on the involvement of PARP and NAD+ in DNA repair processes at the cellular level. However, the degree of NAD+ depletion and the effects of inhibitors are generally unphysiological, and the mechanisms by which niacin deficiency causes pathologies in the whole animal remain uncertain. Given that seven different poly(ADP-ribose) synthesizing enzymes are now known to exist, in addition to numerous mono ADP-ribosyltransferases and enzymes that form cyclic ADP-ribose and NAADP, there are numerous metabolic defects that could be occurring in the niacin-deficient animal. PARP-null mouse models have added confusion to the niacin-deficiency field, in that niacin deficiency can create a catalytically inactive PARP positive environment, in which PARP remains bound to strand breaks and inhibits repair. The poly(ADP-ribose) levels and cancer sensitivity reported in this paper indicate that this process may be occurring in the bone marrow of niacin-deficient rats. This is analogous to the expression of the dominant negative DNA-binding domain of PARP, which has been shown to have severe effects on DNA repair (24
).
Previous work in our laboratory showed that niacin deficiency in rats exacerbates nitrosourea-induced anemia and leukopenia, common acute side effects during chemotherapy (25
). In this study, we showed that niacin deficiency decreases long-term survival after ENU treatment. Use of lower doses of ENU may have allowed a partial mortality and a greater difference between ND and PF rats at the end of the experiment. The extended latency of the niacin-adequate rats could represent many extra years of disease-free life in human populations.
We also showed that niacin deficiency dramatically reduced NAD+ and poly(ADP-ribose) levels in bone marrow. Similar levels of ENU-induced DNA strand breaks in the two dietary groups indicate that metabolism of ENU is not an important factor in the observed differences in cancer progression. The data in this paper support an association of susceptibility to cancer with bone marrow NAD+ and poly(ADP-ribose) levels.
Because the Km of PARP for NAD+ is relatively high (80–130 µmol/L) (7
), and the nucleus cannot compartmentalize NAD+, the argument can be made that poly(ADP-ribose) metabolism is sacrificed during niacin deficiency, whereas redox functions and energy metabolism are maintained (26
). This would be a reasonable strategy for cell survival in the short term, but it would appear to increase the risk of neoplastic transformation in the long term. The results of this study support this conclusion.
There are clear interorgan differences during niacin deficiency, as seen from the comparison of the results of this paper with previous work published in this model. For example, the lung is able to increase NAD+ concentrations in response to chronic oxidant stress, is capable of maintaining this induction during niacin deficiency and is not more sensitive to hyperoxia during niacin deficiency (27
). The liver, which has very high levels of NAD+ and poly(ADP-ribose), even in the deficient state, is not sensitized to the neoplastic effects of DEN by niacin deficiency (28
). In contrast to the liver and lung, the bone marrow is very sensitive to NAD depletion and disruption of poly(ADP-ribose) metabolism, and bone marrow cells are more sensitive to short- and long-term effects of DNA damage in the niacin-deficient state. The rapid turnover and export of cells from the marrow may be the reason for the dramatic decrease in NAD in this tissue. Other tissues with rapid cell division, such as the gastrointestinal tract and skin, may be similarly affected. The occurrence of skin and gastrointestinal symptoms in pellagra patients supports this idea (26
).
DNA damage-induced poly(ADP-ribose) accumulation occurs in a different pattern in the bone marrow than in the liver. When poly(ADP-ribose) levels are measured in the liver after administration of DEN, there is a large increase in polymer accumulation associated primarily with PARP automodification (smear above 116 kDa, Fig. 4
). In the bone marrow, there is an early increase in polymer synthesis on the 116-kDa acceptor 1.5 h after ENU treatment, but 3 h post-ENU, a large portion of the polymer at is attached to a protein of 90 kDa, which is likely the large apoptotic fragment of PARP (29
). Polymer synthesis recedes to basal levels at 12 h, but begins to increase again 24 h post-ENU treatment. The niacin-deficient bone marrow exhibits a dramatically impaired capacity for both basal and ENU-induced poly(ADP-ribose) accumulation. The proteolysis of PARP and a biphasic pattern of poly(ADP-ribose) accumulation suggest the occurrence of apoptosis in the marrow cell population.
There are other acceptors of interest. There is a band, best visible in the DEN-treated liver, 150 kDa, that is often partially obscured by the top of the 116-kDa smear (Fig. 4)
. This could be tankyrase, the telomere-associated PARP, reported to be 142 kDa (30
). Interestingly, modification of this acceptor also appears to be enhanced by DNA damage. There is also a protein of 66 kDa that is modified in liver and bone marrow that shows DNA damage dependence in both tissues. Telomere repeat factor 1 is a substrate for tankyrase and is slightly larger than 66 kDa (30
). This band may also be related to PARP-2 or PARP-3 enzymes. These enzymes are in the 60-kDa range (31
) and may be automodified in response to DNA damage (32
).
Using a conversion factor of 108 cells/mg DNA in rats, previously published data on tissue poly(ADP-ribose) levels may be compared with bone marrow on a per cell basis. Liver and lung have basal poly(ADP-ribose) contents of 250 and 500 fmol/106 cells, respectively (27
,28
), compared with 50 fmol/106 nucleated cells in the marrow. This relatively low level of poly(ADP-ribose) synthesis in the bone marrow may explain why NAD+ depletion did not occur after DNA damage in the bone marrow in these experiments, whereas we have observed NAD+ depletion in the liver after DEN treatment (21
). The intracellular concentration of NAD+ in the bone marrow may be calculated from the average cell volume of 110 fL, obtained from the Coulter channelyzer (data not shown), and estimates of the water content of the cell. The data in this paper suggest a control value of 350 µmol/L, much lower than the quantity found in tissues such as liver, lung, heart and kidney (15
). Bone marrow NAD+ is also more sensitive to depletion during niacin deficiency than any of these other tissues (15
), and these decreases have a greater effect on poly(ADP-ribose) metabolism than that seen in the liver (15
,28
) or lung (27
). The ND bone marrows have an average estimated NAD+ content of 100 µmol/L (calculated using 110 fL cell volume), and the Km of PARP for NAD+ is between 80 and 130 µmol/L (7
). There is a significant impairment of PARP function, beyond the 50% decrease one would expect from a substrate concentration near the Km. This may reflect a greater depletion of cytosolic/nuclear pools, with sequestration of NAD+ to the mitochondria to maintain redox functions (26
).
The similar accumulation of strand breaks in ND and PF 3 h after ENU treatment is an important observation. Although ENU decomposes spontaneously to reactive intermediates that create the DNA lesions, it is possible that the P450 metabolism could increase the clearance rate of the parent compound (33
). If this occurred, it would likely have to be in the liver because there are very low levels of P450 in the bone marrow (34
). If niacin deficiency altered P450 activity, many of our findings could be the result of different levels of DNA damage. Strand breaks were assessed 3 h after ENU treatment, at the time when poly(ADP-ribose) accumulation was maximal, and the results clearly show that the induction of strand breaks is not influenced by niacin deficiency. Previous data from our laboratory also indicate that hepatic P450 metabolism is not affected by niacin deficiency (28
).
Studies performed in cultured cells or in cell-free DNA repair systems have shown that depletion of NAD+ in cultured cells or in vitro systems may inhibit the rejoining of DNA strand breaks (11
,12
,23
). It is thought that the synthesis of poly(ADP-ribose) on PARP is required to create electrostatic repulsion, removing PARP from the strand break and allowing repair to proceed (23
). In our model, 24 h after ENU treatment, the degree of strand break rejoining may be decreased by niacin deficiency (Fig. 6)
. Although intriguing, this result is based on a small number of animals and lacks intermediate time points. We did not continue with this assay for technical reasons (difficulty in processing larger groups of animals), and this hypothesis is now being examined in our laboratory using the comet assay.
In conclusion, the data presented here show that niacin deficiency during exposure to an alkylating agent significantly increases cancer incidence. The changes in cancer frequency correlate with changes in NAD+ and poly(ADP-ribose) levels. Future studies will work toward a model based on current chemotherapy drugs, such as etoposide, which are implicated in the development of secondary cancers (10
).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: BSA, bovine serum albumin; CON, vehicle-gavaged control; DEN, diethylnitrosamine; ENU, ethylnitrosourea; LT20, latency time to 20% morbidity; LT50, latency time to 50% morbidity; ND, niacin deficient; PARP, poly(ADP-ribose) polymerase; PF, pair-fed; ssDNA, single-stranded DNA.
Manuscript received 29 March 2001. Initial review completed 3 May 2001. Revision accepted 1 October 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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