Journal of Perinatology (2016) 36, 132–136 © 2016 Nature America, Inc. All rights reserved 0743-8346/16 www.nature.com/jp

ORIGINAL ARTICLE

Jaundice, phototherapy and DNA damage in full-term neonates N Ramy1, EA Ghany1, W Alsharany1, A Nada2, RK Darwish3, WA Rabie3 and H Aly4 OBJECTIVE: Phototherapy is the standard therapeutic approach for neonatal hyperbilirubinemia. Oxidative effects of phototherapy may have potential harms, including DNA damage. Unconjugated bilirubin (UCB) might also possess antigenotoxic potential. Intensive phototherapy is more efficacious than conventional phototherapy in treating hyperbilirubinemia. This study aimed to assess the impact of hyperbilirubinemia and the two different types of phototherapy on DNA damage in peripheral blood mononuclear cells of neonates. STUDY DESIGN: The study was conducted on term neonates with non-hemolytic hyperbilirubinemia and control healthy neonates. Genotoxicity was assessed using single-cell gel electrophoresis (Comet assay) in peripheral mononuclear cells. Blood samples were obtained at enrollment in all infants and after intensive or conventional phototherapy in jaundiced infants. RESULT: DNA damage did not significantly differ between jaundiced and non-jaundiced neonates (11.4 ± 8.7 and 10.9 ± 8.3 arbitrary units (AU), respectively, P = 0.58). It increased significantly after exposure to phototherapy compared with prephototherapy values (45.6 ± 14.7 vs 11.4 ± 8.7 AU, respectively, P o 0.001). The duration of phototherapy correlated positively with markers of DNA damage (r = 0.86, P o0.001); however, the intensity of used light did not significantly impact genotoxicity. CONCLUSION: Hyperbilirubinemia does not influence DNA damage, whereas both conventional and intensive phototherapy are associated with DNA damage in term infants with hyperbilirubinemia. Journal of Perinatology (2016) 36, 132–136; doi:10.1038/jp.2015.166; published online 19 November 2015

INTRODUCTION Hyperbilirubinemia is a common medical diagnosis in neonates and is the leading cause of hospital readmissions.1 Treatment of unconjugated hyperbilirubinemia primarily relies on the use of phototherapy2; light changes the structure of bilirubin molecule into water-soluble isomers that can be excreted without the need for hepatic conjugation.3 Phototherapy is not known to cause any serious side effects; however, recent animal and cell-culture studies raised concerns regarding its potential damaging effects to DNA.3 Exposure of cells to visible light could induce DNA strand breaks, sister chromatid exchange and mutations.4,5 These DNA breaks cause modifications to DNA that can plausibly lead to diseases related to oxidative stress such as necrotizing enterocolitis and retinopathy of prematurity in newborns and even increase the risk for cancer development in their future life.6 Human cells use many strategies to protect genomic DNA from accumulating such lesions. If the damage is extensive, cell cycle progression is blocked to allow more time for DNA repair. However, if the damage is irreparable the affected cells undergo apoptosis.7 If damages related to reactive oxygen species are not timely repaired, free radicals can cause lipid peroxidation and strand breaks in DNA.8–10 In vitro studies demonstrated the release of long-lived toxic products when bilirubin solutions were exposed to light.5 To assess in vivo DNA damage, the single-cell gel electrophoresis (alkaline Comet assay) is a widely used method. This sensitive test has been used for measurement of the extent of endogenous DNA damage under a variety of conditions.11

In this study, we aimed to clarify the following questions using the alkaline Comet assay: (1) does hyperbilirubinemia and/or phototherapy cause genotoxicity in vivo in full-term newborns? (2) if genotoxic effects occur, is there any relationship between the duration of phototherapy and frequency of genotoxicity? and (3) does the type of phototherapy, whether conventional or intensive, affect genotoxicity?

STUDY METHODS Patients This case–control study was performed in the neonatal intensive care unit of Cairo University Children’s Hospital. A consent was obtained from the parents of each newborn after explaining the whole procedure. The study was approved by the Faculty of Medicine, Cairo University Ethical Committee. The study included neonates who are: (a) full term with gestational age 437 weeks, (b) with postnatal age ⩽ 14 days, (c) presenting with indirect hyperbilirubinemia that required phototherapy as per American Association of Pediatrics (AAP) recommendations, and (d) this hyperbilirubinemia was non-hemolytic as evidenced by absence of blood group iso-immunization, negative Coomb’s test and normal hemoglobin. The study did not include infants with conditions associated with cell damages and genotoxicity and that include infants with metabolic or major congenital anomalies, perinatal asphyxia, sepsis and hemolytic diseases. A control group of apparently healthy full-term infants without clinical jaundice were recruited from the outpatient clinic at Cairo University Children’s Hospital.

1 Pediatrics Department, Faculty of Medicine, Cairo University, Cairo, Egypt; 2Institute of Postgraduate Childhood, Ain Shams University, Cairo, Egypt; 3Clinical and Chemical Pathology Department, Faculty of Medicine, Cairo University, Cairo, Egypt and 4Division of Newborn Services, The George Washington University Medical Center, Washington, DC, USA. Correspondence: Dr H Aly, Division of Newborn Services, The George Washington University Hospital, The George Washington University, 900 23rd Street, NW, Suite G-2092, Room G-132, Washington, DC 20037, USA. E-mail: [email protected] Received 14 July 2015; revised 11 September 2015; accepted 1 October 2015; published online 19 November 2015

Jaundice, phototherapy and DNA damage N Ramy et al

133

Figure 1. Visual analysis. Images of peripheral blood mononuclear cells showing different classes of DNA damage using visual scoring ((a): score = 1, (b): score = 2, (c): score = 3, and (d): score = 4).

Methods Phototherapy. Infants with jaundice were treated with phototherapy based on AAP guidelines.12 Infants received either conventional or intensive forms of light therapy according to corresponding bilirubin concentrations in the serum. Intensive phototherapy was used for infants who were in the high-risk zone of the bilirubin nomogram and was subsequently changed to conventional phototherapy once bilirubin decreased to the intermediate-risk zone.12 Conventional phototherapy systems consisted of six white fluorescent tubes (PhilipsTL52/20W, Buenos Aires, Argentina) placed 40 cm above the infant. Intensive phototherapy systems consisted of 12 white fluorescent tubes (Philips TL03, Ontario, Canada) placed within 20 cm under and above the infant’s front and back. The infants were placed naked, except for a diaper and eye patches, in an incubator or intensive phototherapy unit (Bilicrystal, Medes-time,or Bilisphere 360, Marcinelle, Belgium). The light energy of the phototherapy units was measured using a standard photometer (Light Meter VF, Minolta, Japan); conventional phototherapy units were 12–16 μW cm − 2 nm − 1 and intensive phototherapy units were 30–34 μW cm − 2 nm − 1 in the 430–490nm band. Phototherapy was continuously applied to jaundiced neonates except during feeding and care. Assays. DNA damage was measured in samples drawn from all cases before the start of phototherapy and at the conclusion of the corresponding type of phototherapy. Therefore, infants in the conventional phototherapy group had DNA assays measured before the start and at the conclusion of phototherapy (before discharge from the hospital), whereas infants in the intensive phototherapy group had DNA assays measured before the start of phototherapy and before changing from intensive to conventional phototherapy. Blood samples were collected from a peripheral vein into EDTA vacutainers, stored at 10 °C in the dark to prevent further DNA damage and were processed within 2 h. Mononuclear cell layer was separated on Ficoll hypaque and was first diluted in equal volume of phosphate-buffered saline (PBS) and then layered on the Ficoll and centrifuged at 1800 r.p.m. for 25 min. DNA damage was analyzed with the alkaline Comet assay using the Oxiselect Comet Assay Kit (Cell Biolabs, © 2016 Nature America, Inc.

San Diego, CA, USA). The cells were washed first with ice-cold PBS (without Mg+2 or Ca+2) and re-suspended at 1 × 105 cells ml − 1 in ice-cold PBS. Then cells were combined with comet agarose at 1:10 ratio, mixed and immediately 75 μl well − 1 was pipetted on to one well of the Oxiselect comet slide. Complete well coverage was ensured. The slide was then transferred to a small container containing prechilled lysis buffer and immersed in it overnight in dark. The lysis buffer was then replaced by prechilled alkaline solution for 30 min at 4 °C in the dark. Electrophoresis of slides was carried out in horizontal electrophoresis chamber with cold alkaline solution for 1 h at 300 mA and 1 volt cm − 1. After electrophoresis, the slides were then washed twice by prechilled de-ionized water and then immersed in cold 70% ethanol for 5 min. Then slides were dried up and stained with vista Green DNA dye provided in the kit. Comets were visualized by epifluorescence microscopy using a fluorescein isothiocyanate filter. Images of 100 randomly selected cells from each subject were analyzed visually by an observer who was not aware of the diagnosis. Each image was classified according to the intensity of the fluorescence in the comet tail and was given a value of either 0, 1, 2, 3 or 4 with undamaged class = 0 to maximally damaged class = 4 (Figure 1). The DNA damage was assessed and the score was calculated before and within 24 h after the phototherapy treatment.

Visual analysis There is a scheme for visual scoring based on five recognizable classes of comet, from class 0 (undamaged, no discernible tail) to class 4 (almost all DNA in tail, insignificant head). One hundred comets were selected from each slide avoiding the edges of the gel where high levels of damage are often seen. Each comet is given a value according to the class it is put into, so that an overall score can be derived for each gel, ranging from 0 to 400 arbitrary units (AU) (Figure 1). Slides were visualized by the same investigator who was not aware of the patient groups or the type of treatment received. Journal of Perinatology (2016), 132 – 136

Jaundice, phototherapy and DNA damage N Ramy et al

134 Statistical analysis Data were processed and analyzed using the Statistical Package of Social Science (SPSS) for windows, version 17.0 (SPSS, Chicago, IL, USA). Categorical data were summarized as frequencies (percentages). Associations between nominal data were investigated using Fisher’s exact or Chi square tests. Numerical data were represented as mean and s.d. Differences between groups were detected using Student’s t-test. Pearson’s correlation coefficient was used to test the association between the duration of phototherapy and markers of DNA damage. P-valueo0.05 was considered statistically significant. There were no crossover analyses among groups; therefore data for the conventional phototherapy group represented infants who received only conventional light, whereas data for the intensive phototherapy group were collected only while infants were receiving intensive phototherapy.

Power analysis and sample size calculation Based on a previous study for DNA damage in neonates, to detect a difference of 20% in the frequency of DNA damage between cases and controls, a sample of 60 subjects is required (P = 0.05, power = 90%).13 To detect a difference in the frequency of DNA damage between the intensive and the conventional phototherapy cases that is equal to 1 s.d., a sample of 36 cases is required (P = 0.05, power = 80%).

Table 1.

Demographic data of all infants enrolled in the study

Variables Male/females (number)a Age (days) Birth weight (g) Vaginal/cesarean delivery (number) Gestational age (weeks)

Jaundiced (n = 36)

Control (n = 40)

P-value

19/17 4.69 ± 3.07 3094 ± 292 23/13

19/21 4.77 ± 1.45 3098 ± 282 28/12

0.8 0.9 0.8 0.7

38.3 ± 0.8

38.3 ± 0.9

0.8

Data are presented as numbers or as mean ± s.d.

a

RESULTS The study included 36 newborns; 22 of them received conventional phototherapy and 14 received intensive phototherapy. Forty apparently healthy non-jaundiced full terms were also included as a control group. Demographic data did not differ between groups (n = 76) (Table 1). Routine laboratory examinations were all normal except for greater bilirubin concentrations in jaundiced cases (21.2 ± 4.5 vs 3.77 ± 0.9 mg dl − 1, P o0.0001). There was no difference in bilirubin concentrations between the two phototherapy groups before or after starting treatment (Figure 2). Infants in the conventional group were exposed to phototherapy for a longer duration than the intensive group (62.2 ± 23.02 vs 41.3 ± 22.9 h, P = 0.005). Markers of DNA damage (Comet scores) measured at enrollment did not differ between jaundiced and control infants (P = 0.58; Figure 2). In jaundiced infants, markers of DNA damage significantly increased after exposure to phototherapy (P o 0.001; Figure 2). These markers did not significantly differ between both conventional and intensive light therapy used (P = 0.2; Figure 2). There was a significant positive correlation between the duration of phototherapy and Comet scores (r = 0.86, P o0.001). DISCUSSION We demonstrated that hyperbilirubinemia is not associated with DNA strand breaks, which are a well-known type of DNA damage. Both conventional and intensive phototherapy treatments increased DNA damage. The duration, but not the intensity, of phototherapy correlates with the degree of DNA damage. Although adverse effects of phototherapy have been evaluated clinically in several studies, information on the potential cellular effects of phototherapy such as DNA damage is limited.14 Phototherapy has been widely used to treat infants with jaundice for several decades without noticeable long-term consequences.10 However, damage to DNA is an essential part of genetic toxicology, because chromosomal mutations are considered as

25 p = 0.13

20

10

DNA Damage Arbitrary Unit (AU)

Bilirubin (mg/dL)

30

15 10 5 0

0 Before

Jaundiced

After

Control

75 * p < 0.001

50

25

0

DNA Damage Arbitrary Unit (AU)

75

DNA Damage Arbitrary Unit (AU)

p = 0.58

20

p = 0.2 50

25

0 Before

After

Conventional

Intensive

Figure 2. Bilirubin concentrations and frequency of DNA damage in the study population. (a) Bilirubin concentrations before and after phototherapy in the intensive group (……) and the conventional group (----), (b) DNA damage in cases vs controls (n = 76), (c) significant DNA damage in cases after exposure to phototherapy (n = 36) and (d) DNA damage in cases after receiving conventional and intensive phototherapy (n = 36). Journal of Perinatology (2016), 132 – 136

© 2016 Nature America, Inc.

Jaundice, phototherapy and DNA damage N Ramy et al

135 important events in carcinogenesis. Human peripheral mononuclear leukocytes have been widely used to monitor environmentally induced genetic damage by a variety of methods, such as micronucleus, chromosome aberration and sister chromatid exchange assays.15 We opted to use the single-cell gel electrophoresis (Comet) assay, among the various assays for measuring DNA damage, because it is a sensitive and powerful method for determining DNA strand breaks.16,17 The advantages of this technique include its demonstrated sensitivity for detecting the smallest amount of DNA damage, its feasibility with use of only a few cells per sample and its low cost.18–21 In the current study, bilirubin did not influence DNA damage in the peripheral blood mononuclear cells of full-term infants. There is a debate on the impact of bilirubin on genotoxicity; a previous study reported DNA damage even with low bilirubin concentrations, suggesting possible genotoxic effects of bilirubin in unconjugated hyperbilirubinemia.22 Nevertheless, unconjugated hyperbilirubinemia was shown to associate with oxidative stress in preterm infants, whereas a decrease in bilirubin concentrations corresponded with a decrease in oxidative stress.23,24 On the other hand, other in vitro25,26 and animal27 studies described antioxidant properties of bilirubin. It was shown that bilirubin in micromolar concentrations has stronger antioxidant effects than vitamin E28 and was found to reduce oxidative injury in neonatal rats.27 Even in vivo studies defined bile pigment bilirubin as a known antioxidant. Its protective effect against cancers and cardiovascular diseases might imply bilirubin to possess antigenotoxic properties.29 We demonstrated that phototherapy was associated with increased damage to DNA in infants with hyperbilirubinemia. Genotoxicity increased with the use of both conventional and intensive phototherapies.13 There is evidence that the oxidative effects of phototherapy may harm cell membranes and can potentially lead to lipid peroxidation9 and DNA damage.10 Exposure of cell cultures to phototherapy was associated with DNA strand breaks and mutations.4,5 Membrane-bound bilirubin is a photo-sensitizer that may capture light energy. This photoexcited molecule can subsequently use its energy to convert molecular oxygen into a highly reactive radical.23 Christensen et al.5 demonstrated the phototherapy-induced single-strand breaks in DNA to significantly increase when bilirubin solutions were added to the cells in culture. However, the response to phototherapy in vivo may differ and the chemicals released by exposure to light leading to DNA damage may be too short-lived or their effects may be depressed by other antioxidants in the tissue of newborns.2 In this study, the duration of exposure to phototherapy correlated with the degree of DNA damage. Similar to these findings, previous studies demonstrated increased genotoxicity with longer durations of exposure to light in full-term jaundiced infants.30,31 However, this current study reports a novel finding that the frequency of genotoxicity is not affected by the intensity of phototherapy. We therefore advocate the strategy of using intensive phototherapy for a short period of time; this strategy will be more efficacious and less toxic than using conventional phototherapy for a prolonged time. One of the limitations of this study is that the control group were tested for DNA damage only once. It was not feasible to recall healthy newborns for a second visit to obtain blood samples. Therefore, the study in the current format does not eliminate a theoretical possibility for the effect of time on DNA damage without phototherapy. In addition, this study does not provide follow-up on these infants so as to describe whether phototherapy-induced DNA damage is a transient or a permanent process. In conclusion, this study proves the genotoxic effects of phototherapy. The duration, not the intensity, of phototherapy correlates with the frequency of DNA damage. Hyperbilirubinemia per se, in the absence of hemolysis or sepsis, is not associated with © 2016 Nature America, Inc.

DNA damage in full-term infants. Follow-up studies are needed to determine the course and long-term effects related to DNA damage. It is not clear if these findings are reproducible in the very preterm infants as they are more vulnerable to oxidative stress and they are often exposed to ‘prophylactic’ phototherapy early in life. Therefore, future studies on preterm infants are required. In the meantime, it is wise to avoid unnecessary use of phototherapy in term and preterm infants. CONFLICT OF INTEREST The authors declare no conflict of interest.

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