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A chromosomal-effect study of intensive phototherapy versus conventional phototherapy in newborns with jaundice
Mutation Research 676 (2009) 17–20
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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
A chromosomal-effect study of intensive phototherapy versus conventional phototherapy in newborns with jaundice夽 Ahmet Karadag a,∗ , Ahmet Yesilyurt b , Suna Unal c , Ipek Keskin b , Hilmi Demirin d , Nurdan Uras a , Ugur Dilmen c , M. Mansur Tatli a a
Division of Neonatology, Department of Pediatrics, Fatih University School of Medicine, Ankara, Turkey Department of Clinical Genetics, Zekai Tahir Burak Women’s Health Education and Research Hospital, Ankara, Turkey Division of Neonatology, Zekai Tahir Burak Women’s Health Education and Research Hospital, Ankara, Turkey d Department of Biochemistry, Gülhane Military Medical Academy, Ankara, Turkey b c
a r t i c l e
i n f o
Article history: Received 1 December 2008 Received in revised form 18 February 2009 Accepted 5 March 2009 Available online 17 April 2009 Keywords: Intensive phototherapy Chromosomal side effect Sister chromatid exchange Bilirubin Newborn
a b s t r a c t In this study, we aimed to make a comparison between chromosomal effects caused by conventional phototherapy and intensive phototherapy in jaundiced newborns. The study group included 83 newborns with gestation age of ≥35 weeks, and on days 3–10 after birth. Newborns were divided into four groups on the basis of total serum bilirubin (TSB) levels upon admission and need for phototherapy. The intensive group (n = 19) consisted of newborns who received light-emitting diode (LED) phototherapy, the conventional group (n = 23) consisted of newborns who received conventional phototherapy, the jaundiced control group (n = 21) consisted of newborns whose TSB levels were higher than 10 mg/dL (average = 13.7 ± 1.5 mg/dL) on admission and who did not receive phototherapy, and the non-jaundiced control group (n = 20) consisted of newborns whose TSB levels were less than 5 mg/dl (average = 3.6 ± 0.8 mg/dL). TSB level of the intensive group at admission was 20.2 ± 1.3 mg/dL, whereas the level of conventional group was 19.6 ± 1.5 mg/dL. Blood samples were taken from all infants on admission to determine sister chromatid exchange (SCE1 ) frequency. Blood sampling was repeated on discharge (SCE2 ) of infants who had received phototherapy. Demographic information, hospitalization details and the rate of decline in TSB were recorded, and frequencies of SCE1 and SCE2 were compared. There was no difference in demographic information among the four groups. SCE1 frequencies in 50 metaphases were evaluated in the intensive, conventional, jaundiced control and non-jaundiced control groups, and the SCE1 frequency was determined as 9.37/cell, 9.54/cell, 9.23/cell and 6.17/cell, respectively. The SCE1 frequency of the jaundiced groups (intensive, conventional and newborns-with-jaundice control group) was significantly higher than that in the non-jaundiced control group (p = 0.001). There was no significant difference between the intensive group and the conventional group in SCE2 frequency (13.5/cell vs 13.55/cell, p = 0.39). SCE2 frequency was higher than SCE1 frequency in both the intensive and conventional groups (p = 0.001). A strong correlation was found between admission TSB and SCE1 frequency (p = 0.001; r = 0.79). The rate of decline in TSB was higher in the intensive group compared with the conventional group (0.26 mg/(dL h) vs 0.14 mg/(dL h); p = 0.001). We found that intensive and conventional phototherapies similarly increase SCE frequency in newborns. There was a strong, positive correlation between the TSB-on-admission level and SCE1 frequency. In the light of this study, we may conclude that intensive and conventional phototherapies may have an effect on chromosomes in jaundiced newborns. TSB levels higher than 10 mg/dL are, too, reported hazardous on chromosomes. Further studies are warranted to elucidate this relationship. © 2009 Elsevier B.V. All rights reserved.
1. Introduction
夽 This study was supported by grant from the Fatih University School of Medicine Project Center (Project Number: P-53010803-2). ∗ Corresponding author at: Division of Neonatology, Department of Pediatrics, Fatih University School of Medicine, Hosdere Cad. No: 145, 06540 Cankaya, Ankara, Turkey. Tel.: +90 506 336 74 80; fax: +90 312 441 54 98. E-mail address: [email protected] (A. Karadag). 1383-5718/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2009.03.008
Although phototherapy has been used for the treatment of neonatal jaundice for more than 50 years, the most efficacious phototherapy method with the least side effects has not been developed yet. The reported side effects of phototherapy have been subject to extensive and controversial debate, and include rashes, loose green stool, water loss, oxidative injury, and dehydration [1,2]. However, chromosomal side effects of the phototherapy began to be reported
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A. Karadag et al. / Mutation Research 676 (2009) 17–20
in some recent experimental studies [3,4]. DNA damage caused by phototherapy in jaundiced newborns has been, thereafter, shown in clinical studies [5,6]. Efficacy of phototherapy is dependent on the colour (wavelength) and intensity (irradiance) of the light emitted during phototherapy, the exposed body surface area, and the duration of exposure [2]. The American Academy of Pediatrics (AAP) defines intensive phototherapy as irradiance of at least 30 mW/(cm2 nm) in the 430–490 nm band, and suggests intensive phototherapy to treat neonatal jaundice for gaining a faster bilirubin decrease [7]. Recently, high-intensity gallium nitride light-emitting diodes (LEDs) have been developed and studied as possible light sources for intensive phototherapy of neonatal jaundice [8]. Blue LEDs emit a high-intensity narrow band of blue light overlapping the peak spectrum of bilirubin breakdown [8], resulting in potentially shorter treatment times [9]. LEDs are also power-efficient, they are light in weight, produce less heat, and have a longer lifetime [8,10]. Although efficacy studies about the LEDs were performed, and the technique is being used much more than ever before in neonatology services, there is no study investigating whether there are chromosomal side effects in jaundiced newborns. There are studies concerning side effects of conventional phototherapy on either DNA or chromosomes. Sister chromatid exchange (SCE) is a cytogenetic indicator arising during replication of damaged DNA templates from reciprocal DNA interchanges between sister chromatids during the replication process [11]. SCEs can arise from DNA damage occurring before DNA replication [12]. They can occur at certain rates normally, and as a consequence of exposure to ultraviolet light, ionizing radiation, environmental hazards, chemotherapeutics, viral infections, psoralens-plus-ultraviolet A (PUVA) therapy, and in malignancies [13]. We investigated in a clinical study using SCE analysis whether there are any chromosomal side effects of conventional and/or intensive phototherapy. 2. Materials and methods This study was conducted between January 2008 and May 2008 on neonates with jaundice admitted to two neonatal units of different centers in Turkey. The study protocol was approved by the Ethics Committee of each Institute. Informed consent was obtained from all parents of the newborns involved in the study. 2.1. Patient selection The study group included 83 newborns with gestation age of ≥35 weeks and on days 3–10 after birth. Patients were divided into four groups on the basis of the total bilirubin levels in serum (TSB) upon admission, and the need for phototherapy. The groups were formed as: an intensive group who needed phototherapy according to the suggestions of the AAP, and received intensive phototherapy (n = 19). The conventional group received conventional phototherapy (n = 23) [7]. We designed two different control groups in order both to make a comparison between the two therapy groups, and to investigate any chromosomal side effects of serum bilirubin independent of phototherapy. The infants with jaundice (TSB level ≥10 mg/dL) but excluded from phototherapy because the levels were below the AAP suggestions were placed in the jaundiced control group (n = 21); the ones with no apparent jaundice (TSB level ≤5 mg/dL) were assigned to the non-jaundiced control group (n = 20). Newborns with a gestational age of <35 weeks or >42 weeks were defined as pre-term and post-term, respectively, and were not included in the study. Other exclusion criteria were a smaller or larger body weight than normal for gestational age, determined on the basis of the Colorado intrauterine growth charts [14], direct bilirubinemia, multiple gestation, any systemic disease, neonates that needed phototherapy within the first 3 days of life, any congenital malformation, respiratory distress, glucose-6-phosphate dehydrogenase deficiency, clinical- or culture-proven sepsis, and inability to initiate or maintain oral feedings within 3 h after birth due to various reasons. 2.2. Phototherapy Phototherapy was applied to the intensive group with Neoblue® LED phototherapy system (Natus Medical Inc. San Carlos, CA, USA, intensity = 35 W/(cm2 nm), spectrum 450–470 nm). An Elektro-Mag® M304, Philips TL 20W/52 with a pair of
blue-ray fluorescent lamps, intensity = 10 W/(cm2 nm) and spectrum 450–560 nm was used in the conventional group. Before starting phototherapy on a subject, the irradiance was checked with a photoradiometer (Fluoro-lite 451® , Minolta/Air Shields, USA). Our aim was to maintain the irradiance above 35 W/(nm cm2 ) during intensive phototherapy and above 10 W/(nm cm2 ) in the conventional group. The irradiance of the lamps was measured weekly, and if decreased, lamps were replaced whenever necessary, to maintain this irradiance. All infants were exposed, completely unclothed with their eyes and genitals covered, to continuous phototherapy that was interrupted only for feeding, cleaning and blood sampling. The infants’ weights and temperatures were monitored. Gestational ages, types of feeding, ages at phototherapy, possible side effects concerning the phototherapy (weight loss, rashes, diarrhea, and thermal changes), TSB level at initiation of phototherapy and at termination of phototherapy, as well as duration of phototherapy of all the subjects were recorded. Randomization in phototherapy groups was maintained according to admission to the hospital. The first patient was included in the conventional group, and the other ones were placed in either group alternately in order. The phototherapy was stopped, as recommended by the AAP, when the TSB decreased to a level of at least 2 mg/dL below the suggested AAP guideline, unique for each patient. 2.3. Blood samples The first blood samples of the newborns involved in the study were taken for phenylketonuria screening, Guthrie’s Test, within 3–10 days after birth when they entered the newborn outpatient clinic. Peripheral veins were used and a 0.5-mL blood sample for TSB analyses, and another 0.5 mL for pre-treatment SCE frequency (SCE1 ) were collected. The samples for SCE1 were transferred to the genetics lab within 1 h after collection in heparinized pediatric tubes. The routine serum tubes for TSB analyses were covered with aluminum foil to avoid exposure to sunlight, and immediately sent to the labs for analysis. TSB levels of subjects in phototherapy groups were checked every 12 h after hospitalization. The last sample in which the TSB had decreased to a level of 2 mg/dL below the suggested AAP guideline was conveyed at room temperature to the genetics lab within max 1 h in order to analyze the frequency of SCE (SCE2 ). SCE2 analysis was not carried out in the jaundiced and non-jaundiced control groups. 2.4. Analysis of sister chromatid exchange For SCE analysis, 0.5 mL of heparinized blood was drawn from each individual. Cultures were established by adding 0.25 mL of blood to 5 mL karyotyping medium (Biological Industries, Beit Haemek, Israel) with 2% phytohaemagglutinin M (PHA) (Biological Industries, Beit Haemek, Israel), and incubating for 24 h at 37 ◦ C. A 5-bromo-2 -deoxyuridine (BrdU) (Sigma, St. Louis, MO, USA) solution was added to a final concentration of 5 g/mL. Lymphocytes were cultured in the dark for 48 h and metaphases were blocked during the last 2 h with Colcemid (Biological Industries, Beit Haemek, Israel) at a final concentration of 0.1 g/mL. Further processing included hypotonic treatment, fixation, slide preparation and Fluorescein plus Giemsa (FPG) staining for the detection of SCE [15]. Fifty second-division metaphases were scored on coded slides by a single observer, and expressed as the number of SCEs/cell per subject. Staffs performing the SCE analysis were blinded to the study. 2.5. Statistics SPPS 13.0 for Windows® (SPSS Inc., Chicago, IL) was used for statistics in the study. A p-value of <0.05 was considered as statistically significant. The convenience to normal distribution in continuous variables was analyzed by Kolmogorov–Smirnov’s Test of One Sample at the end of which the results were negative, and analysis was continued with non-parametric tests. Results are expressed as means in descriptive statistics with regard to the normal distributed variables. The Kruskal–Wallis and Mann–Whitney U-tests were used to determine significance. Pearson’s Correlation Analysis was used for the assessment of relation between the parametric variables. A Wilcoxon’s Rank Test was performed for the assessment of any difference between the first and second values of the dependent groups.
3. Results Eighty-three newborns were involved in the study, 19 of whom were in the intensive group, 23 in the conventional, 21 in the jaundiced control group, and 20 in the non-jaundiced controls. The groups analyzed did not show statistically significant differences in terms of birth weight, age and weight at the moment of application, route of delivery, and type of feeding. TSB levels differed significantly as expected. No difference was seen between intensive and conventional phototherapy groups (p = 0.4); the TSB levels of these groups were higher than those of the two control
A. Karadag et al. / Mutation Research 676 (2009) 17–20
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Table 1 Baseline demographic data of newborns in the study.
Male/female Gestational age (week) (mean ± SD) Birth weight (g) (mean ± SD) Vaginal/cesarean delivery (%) Admission weight (g) (mean ± SD) Admission age (h) (mean ± SD) Admission TSB level (mg/dL) (mean ± SD)
Intensive phototherapy, n = 19
Conventional phototherapy, n = 23
Jaundiced control, n = 21
Non-jaundiced control, n = 20
pa
11/8 37.8 ± 1.2 3288 ± 493 26.3/73.7 3133 ± 525 111.5 ± 28.1 20.2 ± 1.3
11/12 38.6 ± 1.8 3236 ± 522 35/65 3082 ± 512 114.1 ± 27.7 19.6 ± 1.5
12/9 38.4 ± 1.5 3317 ± 373 26.1/73.9 3168 ± 401 96.2 ± 18.3 13.7 ± 1.5
9/11 38.4 ± 1.8 3219 ± 399 14.3/85.7 3219 ± 399 101.8 ± 25.1 3.6 ± 0.8
0.133 0.23 0.83 0.78 0.424 0.65 0.001b
TSB: total serum bilirubin. a p-Value resembles all groups. b There is no difference between phototherapy groups, and TSB levels are higher than in the two control groups. Jaundiced control group has significantly higher TSB levels than non-jaundiced control group.
groups (p = 0.001). Also, TSB levels of the jaundiced control group were significantly higher than those of the non-jaundiced control group (p = 0.001). Baseline characteristics of the participants were summarized in Table 1. No difference was found between the two phototherapy groups in terms of side effects possibly related to the phototherapy, like weight loss, alterations in body temperature, dermal reactions and daily number of defecations. There was no significant difference between the two phototherapy groups in terms of hemoglobin, hematocrit and TSB levels at the time of application and discharge. However, a significant difference was recorded with regards to phototherapy duration and rate of decrease in bilirubin levels (Table 2), the average duration of treatment in the intensive group being shorter (29.9 ± 11.3 h vs 46.0 ± 14.1 h; p = 0.001), and the rate of decrease in bilirubin per hour being greater (0.26 ± 0.07 mg/(dL h) vs 0.14 ± 0.04 mg/(dL h); p = 0.001). When we take notice of SCE1 , the intensive, conventional and jaundiced controls did not show any difference, whereas these three groups showed an evident increase in SCE compared with the non-jaundiced group (p = 0.001) (Table 3). SCE2 frequency was slightly higher in the intensive group compared with the conventional group, but this was not significant (p = 0.39). SCE1 frequencies were significantly lower than SCE2 frequencies in both phototherapy groups (p = 0.001, p = 0.001, respectively). Neither group had chromosome fragmentation. Correlation analysis made on the variables affecting the SCE1 frequency showed a strong correlation between TSB levels on admission and SCE1 frequency (p = 0.001; r = 0.87) (Fig. 1).
Table 2 Comparison of phototherapy groups. Intensive phototherapy, n = 19 Admission TSB (mg/dL) Discharge TSB (mg/dL) Hemoglobin (g/dL) Hematocrit (%) Phototherapy duration (h) TSB lowering rate (mg/(dL h))
20.2 12.9 17.6 50.8 29.9 0.26
± ± ± ± ± ±
1.3 1.6 1.7 5.6 11.3 0.07
Conventional phototherapy, n = 23 19.6 13.2 17.1 51.6 46.0 0.14
± ± ± ± ± ±
1.5 1.3 1.5 5.1 14.1 0.04
p
0.09 0.58 0.47 0.72 0.001 0.001
TSB: total serum bilirubin.
Fig. 1. The correlation of SCE1 frequencies and admission TSB levels.
4. Discussion We found that both the intensive and the conventional therapies induce an increase in SCE frequencies of T-lymphocytes of newborns during the period of treatment. Moreover, TSB levels >10 mg/dL increased SCE frequencies as well. In fact, phototherapy and bilirubin as a photosynthesizing agent have been known for a long time to have some harmful effects on DNA [4–6,16]. Christensen et al. showed that less DNA damage and cytotoxicity were found in cell cultures after irradiation with green light than after blue light [17]. Wu et al. exposed G0 lymphocytes isolated from newborns to blue or green phototherapy light and found that both colours, but especially blue light, induced SCE [18]. SCE frequency is a more sensitive biomarker of chromosomal changes because of its sensitivity to low levels of genotoxic agents [15,19]. We, too, used the SCE method in this study in order to assess chromosomal change. The first clinical study that showed phototherapy to increase SCE frequency was published by GoyanesVillaescusa et al. who compared SCE frequencies in T-lymphocytes of newborns in three groups of phototherapy, non-phototherapy
Table 3 SCE frequencies in the groups during the study.
SCE1 SCE2
Intensive phototherapy n = 19
Conventional phototherapy n = 23
Jaundiced control n = 21
Non-jaundiced control n = 20
p
9.37 13.5
9.74 13.55
9.23 –
6.17 –
0.001a 0.39
SCE: sister chromatid exchange. a p = 0.001: versus non-jaundiced control.
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but jaundiced, and non-jaundiced [20]. Frequencies were significantly higher in newborns of phototherapy group than in controls. Later, Schwartz et al. reported no difference in terms of SCE frequencies between newborns of phototherapy and control groups [21]. Another study, too, found a similar result [22]. These three studies had fewer participants than ours. Furthermore, gestational ages and application ages were not homogenous, phototherapy starting and cessation indications were not standardized, and, probably most important, only conventional therapy was used in these studies. For that reason our study seems to be the first clinical study investigating chromosomal side effects of intensive phototherapy. We had also shown, by means of the comet assay in a previous study, that conventional phototherapy may have harmful effects on DNA [5], but since only conventional therapy was used we could not conclude whether intensive therapy has chromosomal side effects. Also few data are present in the literature about extrachromosomal effects of intensive therapy. LED phototherapy is reported to increase trans-epidermal water loss to a similar extent as conventional phototherapy [23] and to cause apoptosis in rat intestine much more than does conventional therapy [24]. Yet we could not find any general side effects probably related to the phototherapy in our study. It has been known for a long time that stray light has a decreasing effect on bilirubin levels in neonatal jaundice [25]. In addition, we know that this happens due to the photosynthesizing nature of the specific wavelength on bilirubin [17]. We paid attention to this aspect by shielding the specimens in order to prevent our results from being affected by the stray light. But still we were unable to strictly standardize the conditions since technical problems necessarily emerged, due to the lights in the room or the day–light the patients were exposed to, which could have an influence on the decrease of the bilirubin and SCE levels. However, we ignored this minor effect in the study. One of the additional results of our study was the comparison of the effectiveness of the two therapies, in that the intensive therapy was found more effective in the treatment of jaundice of the newborn. The rate of bilirubin decreased per hour was apparently higher in the intensive therapy group, and the hospitalization duration was shorter. The latter was in concordance with the literature [10,26]. The two most important factors to affect this decrease arise from the overt difference in irradiance between the methods, chosen as approximately 35 W/(cm2 nm) in intensive therapy, and 10 W/(cm2 nm) in the conventional phototherapy. A difference in SCE frequencies was suspected in our study because of this apparent distinction, but the results were nearly the same. We considered the shorter therapy period in the intensive therapy probably accounting for this similarity, despite the stronger irradiance. Although this study was not a longitudinal investigation of longterm effects of phototherapy, its results suggest that the kind of phototherapy widely used in neonatology units can increase chromosomal effects in newborns. In this study we demonstrated that the LED technology and the conventional phototherapy caused a similar increase in SCE frequency in newborns, but no clear chromosomal fragmentations in any of the patients were seen. Higher SCE frequencies were seen in newborns whose TSB was >10 mg/dL than in those with >5 mg/dL, as a result of which bilirubin, independently, was supposed to be a possibly toxic agent to chromosomes. Notwithstanding these data and those in the literature supporting chromosomal side effects of phototherapy, none is serious enough to constitute a drawback for its usage in newborn’s jaundice. There is no evidence that the chromosomal side effect of the phototherapy is permanent and that it causes chronic diseases or cancer in further years. For that reason, wider, randomized and controlled clinical studies investigating the relation between phototherapy and chromosomal side effects are needed. Given the incomplete understanding of these problems, the safest approach
at this time is to restrict the use of phototherapy to cases of serious neonatal jaundice. In this respect, the higher bilirubin threshold for starting phototherapy recommended in the AAP guideline is important. Conflict of interest There are no conflicts of interest. References [1] H.J. Vreman, R.J. Wong, D.K. Stevenson, Phototherapy: current methods and future directions, Semin. Perinatol. 28 (2004) 326–333. [2] M.J. Maisels, A.F. McDonagh, Phototherapy for neonatal jaundice, N. Engl. J. Med. 358 (2008) 920–928. [3] E.B. Roll, T. Christensen, Formation of photoproducts and cytotoxicity of bilirubin irradiated with turquoise and blue phototherapy light, Acta Paediatr. 94 (2005) 1448–1454. [4] B.S. Rosenstein, J.M. Ducore, Enhancement by bilirubin of DNA damage induced in human cells exposed to phototherapy light, Pediatr. Res. 18 (1984) 3– 6. [5] M.M. Tatli, C. Minnet, A. Kocyigit, A. Karadag, Phototherapy increases DNA damage in lymphocytes of hyperbilirubinemic neonates, Mutat. Res. 654 (2008) 93–95. [6] A. Aycicek, A. Kocyigit, O. Erel, H. Senturk, Phototherapy causes DNA damage in peripheral mononuclear leukocytes in term infants, J. Pediatr. (Rio J) 84 (2008) 141–146. [7] Management of hyperbilirubinemia in the newborn infant 35 or more weeks of gestation, Pediatrics 114 (2004) 297–316. [8] H.J. Vreman, R.J. Wong, D.K. Stevenson, R.K. Route, S.D. Reader, M.M. Fejer, R. Gale, D.S. Seidman, Light-emitting diodes: a novel light source for phototherapy, Pediatr. Res. 44 (1998) 804–809. [9] J.F. Ennever, Blue light, green light, white light, more light: treatment of neonatal jaundice, Clin. Perinatol. 17 (1990) 467–481. [10] D.S. Seidman, J. Moise, Z. Ergaz, A. Laor, H.J. Vreman, D.K. Stevenson, R. Gale, A new blue light-emitting phototherapy device: a prospective randomized controlled study, J. Pediatr. 136 (2000) 771–774. [11] J.D. Tucker, R.J. Preston, Chromosome aberrations, micronuclei, aneuploidy, sister chromatid exchanges, and cancer risk assessment, Mutat. Res. 365 (1996) 147–159. [12] G. Renault, A. Gentil, I. Chouroulinkov, Kinetics of induction of sister-chromatid exchanges by X-rays through two cell cycles, Mutat. Res. 94 (1982) 359– 368. [13] D.M. Wilson, L.H. Thompson 3rd, Molecular mechanisms of sister-chromatid exchange, Mutat. Res. 616 (2007) 11–23. [14] L.O. Lubchenco, C. Hansman, E. Boyd, Intrauterine growth in length and head circumference as estimated from live births at gestational ages from 26 to 42 weeks, Pediatrics 37 (1966) 403–408. [15] S.A. Latt, R.R. Schreck, Sister chromatid exchange analysis, Am. J. Hum. Genet. 32 (1980) 297–313. [16] W.T. Speck, H.S. Rosenkranz, Phototherapy for neonatal hyperbilirubinemia—a potential environmental health hazard to newborn infants: a review, Environ. Mutagen. 1 (1979) 321–336. [17] T. Christensen, G. Kinn, T. Granli, I. Amundsen, Cells, bilirubin and light: formation of bilirubin photoproducts and cellular damage at defined wavelengths, Acta Paediatr. 83 (1994) 7–12. [18] F.Y. Wu, K. Iijima, D. Takiguchi, A. Nishida, M. Higurashi, Effect of phototherapy on sister-chromatid exchange in infants with Down syndrome, Mutat. Res. 283 (1992) 65–67. [19] E. Schmid, M. Bauchinger, S. Pietruck, G. Hall, Cytogenetic action of lead in human peripheral lymphocytes in vitro and in vivo, Mutat. Res. 16 (1972) 401–406. [20] V.J. Goyanes-Villaescusa, M. Ugarte, A. Vazquez, Sister chromatid exchange in babies treated by phototherapy, Lancet 2 (1977) 1084–1085. [21] A.L. Schwartz, F.S. Cole, F. Fiedorek, D. Matthews, I. Paika, I.D. Frantz, S.A. Latt, Effect of phototherapy on sister chromatid exchange in premature infants, Lancet 2 (1978) 157–158. [22] N.H. Hatcher, H.M. Risemberg, M.M. Powers, E.B. Hook, Sister-chromatid exchange and phototherapy, Mutat. Res. 60 (1979) 401–403. [23] G. Bertini, S. Perugi, S. Elia, S. Pratesi, C. Dani, F.F. Rubaltelli, Transepidermal water loss and cerebral hemodynamics in preterm infants: conventional versus LED phototherapy, Eur. J. Pediatr. 167 (2008) 37–42. [24] K. Tanaka, H. Hashimoto, T. Tachibana, H. Ishikawa, T. Ohki, Apoptosis in the small intestine of neonatal rat using blue light-emitting diode devices and conventional halogen–quartz devices in phototherapy, Pediatr. Surg. Int. 24 (2008) 837–842. [25] R.J. Cremer, P.W. Perryman, D.H. Richards, Influence of light on the hyperbilirubinaemia of infants, Lancet 1 (1958) 1094–1097. [26] Y.S. Chang, J.H. Hwang, H.N. Kwon, C.W. Choi, S.Y. Ko, W.S. Park, S.M. Shin, M. Lee, In vitro and in vivo efficacy of new blue light emitting diode phototherapy compared to conventional halogen quartz phototherapy for neonatal jaundice, J. Korean Med. Sci. 20 (2005) 61–64.
Contents lists available at ScienceDirect
Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
A chromosomal-effect study of intensive phototherapy versus conventional phototherapy in newborns with jaundice夽 Ahmet Karadag a,∗ , Ahmet Yesilyurt b , Suna Unal c , Ipek Keskin b , Hilmi Demirin d , Nurdan Uras a , Ugur Dilmen c , M. Mansur Tatli a a
Division of Neonatology, Department of Pediatrics, Fatih University School of Medicine, Ankara, Turkey Department of Clinical Genetics, Zekai Tahir Burak Women’s Health Education and Research Hospital, Ankara, Turkey Division of Neonatology, Zekai Tahir Burak Women’s Health Education and Research Hospital, Ankara, Turkey d Department of Biochemistry, Gülhane Military Medical Academy, Ankara, Turkey b c
a r t i c l e
i n f o
Article history: Received 1 December 2008 Received in revised form 18 February 2009 Accepted 5 March 2009 Available online 17 April 2009 Keywords: Intensive phototherapy Chromosomal side effect Sister chromatid exchange Bilirubin Newborn
a b s t r a c t In this study, we aimed to make a comparison between chromosomal effects caused by conventional phototherapy and intensive phototherapy in jaundiced newborns. The study group included 83 newborns with gestation age of ≥35 weeks, and on days 3–10 after birth. Newborns were divided into four groups on the basis of total serum bilirubin (TSB) levels upon admission and need for phototherapy. The intensive group (n = 19) consisted of newborns who received light-emitting diode (LED) phototherapy, the conventional group (n = 23) consisted of newborns who received conventional phototherapy, the jaundiced control group (n = 21) consisted of newborns whose TSB levels were higher than 10 mg/dL (average = 13.7 ± 1.5 mg/dL) on admission and who did not receive phototherapy, and the non-jaundiced control group (n = 20) consisted of newborns whose TSB levels were less than 5 mg/dl (average = 3.6 ± 0.8 mg/dL). TSB level of the intensive group at admission was 20.2 ± 1.3 mg/dL, whereas the level of conventional group was 19.6 ± 1.5 mg/dL. Blood samples were taken from all infants on admission to determine sister chromatid exchange (SCE1 ) frequency. Blood sampling was repeated on discharge (SCE2 ) of infants who had received phototherapy. Demographic information, hospitalization details and the rate of decline in TSB were recorded, and frequencies of SCE1 and SCE2 were compared. There was no difference in demographic information among the four groups. SCE1 frequencies in 50 metaphases were evaluated in the intensive, conventional, jaundiced control and non-jaundiced control groups, and the SCE1 frequency was determined as 9.37/cell, 9.54/cell, 9.23/cell and 6.17/cell, respectively. The SCE1 frequency of the jaundiced groups (intensive, conventional and newborns-with-jaundice control group) was significantly higher than that in the non-jaundiced control group (p = 0.001). There was no significant difference between the intensive group and the conventional group in SCE2 frequency (13.5/cell vs 13.55/cell, p = 0.39). SCE2 frequency was higher than SCE1 frequency in both the intensive and conventional groups (p = 0.001). A strong correlation was found between admission TSB and SCE1 frequency (p = 0.001; r = 0.79). The rate of decline in TSB was higher in the intensive group compared with the conventional group (0.26 mg/(dL h) vs 0.14 mg/(dL h); p = 0.001). We found that intensive and conventional phototherapies similarly increase SCE frequency in newborns. There was a strong, positive correlation between the TSB-on-admission level and SCE1 frequency. In the light of this study, we may conclude that intensive and conventional phototherapies may have an effect on chromosomes in jaundiced newborns. TSB levels higher than 10 mg/dL are, too, reported hazardous on chromosomes. Further studies are warranted to elucidate this relationship. © 2009 Elsevier B.V. All rights reserved.
1. Introduction
夽 This study was supported by grant from the Fatih University School of Medicine Project Center (Project Number: P-53010803-2). ∗ Corresponding author at: Division of Neonatology, Department of Pediatrics, Fatih University School of Medicine, Hosdere Cad. No: 145, 06540 Cankaya, Ankara, Turkey. Tel.: +90 506 336 74 80; fax: +90 312 441 54 98. E-mail address: [email protected] (A. Karadag). 1383-5718/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2009.03.008
Although phototherapy has been used for the treatment of neonatal jaundice for more than 50 years, the most efficacious phototherapy method with the least side effects has not been developed yet. The reported side effects of phototherapy have been subject to extensive and controversial debate, and include rashes, loose green stool, water loss, oxidative injury, and dehydration [1,2]. However, chromosomal side effects of the phototherapy began to be reported
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in some recent experimental studies [3,4]. DNA damage caused by phototherapy in jaundiced newborns has been, thereafter, shown in clinical studies [5,6]. Efficacy of phototherapy is dependent on the colour (wavelength) and intensity (irradiance) of the light emitted during phototherapy, the exposed body surface area, and the duration of exposure [2]. The American Academy of Pediatrics (AAP) defines intensive phototherapy as irradiance of at least 30 mW/(cm2 nm) in the 430–490 nm band, and suggests intensive phototherapy to treat neonatal jaundice for gaining a faster bilirubin decrease [7]. Recently, high-intensity gallium nitride light-emitting diodes (LEDs) have been developed and studied as possible light sources for intensive phototherapy of neonatal jaundice [8]. Blue LEDs emit a high-intensity narrow band of blue light overlapping the peak spectrum of bilirubin breakdown [8], resulting in potentially shorter treatment times [9]. LEDs are also power-efficient, they are light in weight, produce less heat, and have a longer lifetime [8,10]. Although efficacy studies about the LEDs were performed, and the technique is being used much more than ever before in neonatology services, there is no study investigating whether there are chromosomal side effects in jaundiced newborns. There are studies concerning side effects of conventional phototherapy on either DNA or chromosomes. Sister chromatid exchange (SCE) is a cytogenetic indicator arising during replication of damaged DNA templates from reciprocal DNA interchanges between sister chromatids during the replication process [11]. SCEs can arise from DNA damage occurring before DNA replication [12]. They can occur at certain rates normally, and as a consequence of exposure to ultraviolet light, ionizing radiation, environmental hazards, chemotherapeutics, viral infections, psoralens-plus-ultraviolet A (PUVA) therapy, and in malignancies [13]. We investigated in a clinical study using SCE analysis whether there are any chromosomal side effects of conventional and/or intensive phototherapy. 2. Materials and methods This study was conducted between January 2008 and May 2008 on neonates with jaundice admitted to two neonatal units of different centers in Turkey. The study protocol was approved by the Ethics Committee of each Institute. Informed consent was obtained from all parents of the newborns involved in the study. 2.1. Patient selection The study group included 83 newborns with gestation age of ≥35 weeks and on days 3–10 after birth. Patients were divided into four groups on the basis of the total bilirubin levels in serum (TSB) upon admission, and the need for phototherapy. The groups were formed as: an intensive group who needed phototherapy according to the suggestions of the AAP, and received intensive phototherapy (n = 19). The conventional group received conventional phototherapy (n = 23) [7]. We designed two different control groups in order both to make a comparison between the two therapy groups, and to investigate any chromosomal side effects of serum bilirubin independent of phototherapy. The infants with jaundice (TSB level ≥10 mg/dL) but excluded from phototherapy because the levels were below the AAP suggestions were placed in the jaundiced control group (n = 21); the ones with no apparent jaundice (TSB level ≤5 mg/dL) were assigned to the non-jaundiced control group (n = 20). Newborns with a gestational age of <35 weeks or >42 weeks were defined as pre-term and post-term, respectively, and were not included in the study. Other exclusion criteria were a smaller or larger body weight than normal for gestational age, determined on the basis of the Colorado intrauterine growth charts [14], direct bilirubinemia, multiple gestation, any systemic disease, neonates that needed phototherapy within the first 3 days of life, any congenital malformation, respiratory distress, glucose-6-phosphate dehydrogenase deficiency, clinical- or culture-proven sepsis, and inability to initiate or maintain oral feedings within 3 h after birth due to various reasons. 2.2. Phototherapy Phototherapy was applied to the intensive group with Neoblue® LED phototherapy system (Natus Medical Inc. San Carlos, CA, USA, intensity = 35 W/(cm2 nm), spectrum 450–470 nm). An Elektro-Mag® M304, Philips TL 20W/52 with a pair of
blue-ray fluorescent lamps, intensity = 10 W/(cm2 nm) and spectrum 450–560 nm was used in the conventional group. Before starting phototherapy on a subject, the irradiance was checked with a photoradiometer (Fluoro-lite 451® , Minolta/Air Shields, USA). Our aim was to maintain the irradiance above 35 W/(nm cm2 ) during intensive phototherapy and above 10 W/(nm cm2 ) in the conventional group. The irradiance of the lamps was measured weekly, and if decreased, lamps were replaced whenever necessary, to maintain this irradiance. All infants were exposed, completely unclothed with their eyes and genitals covered, to continuous phototherapy that was interrupted only for feeding, cleaning and blood sampling. The infants’ weights and temperatures were monitored. Gestational ages, types of feeding, ages at phototherapy, possible side effects concerning the phototherapy (weight loss, rashes, diarrhea, and thermal changes), TSB level at initiation of phototherapy and at termination of phototherapy, as well as duration of phototherapy of all the subjects were recorded. Randomization in phototherapy groups was maintained according to admission to the hospital. The first patient was included in the conventional group, and the other ones were placed in either group alternately in order. The phototherapy was stopped, as recommended by the AAP, when the TSB decreased to a level of at least 2 mg/dL below the suggested AAP guideline, unique for each patient. 2.3. Blood samples The first blood samples of the newborns involved in the study were taken for phenylketonuria screening, Guthrie’s Test, within 3–10 days after birth when they entered the newborn outpatient clinic. Peripheral veins were used and a 0.5-mL blood sample for TSB analyses, and another 0.5 mL for pre-treatment SCE frequency (SCE1 ) were collected. The samples for SCE1 were transferred to the genetics lab within 1 h after collection in heparinized pediatric tubes. The routine serum tubes for TSB analyses were covered with aluminum foil to avoid exposure to sunlight, and immediately sent to the labs for analysis. TSB levels of subjects in phototherapy groups were checked every 12 h after hospitalization. The last sample in which the TSB had decreased to a level of 2 mg/dL below the suggested AAP guideline was conveyed at room temperature to the genetics lab within max 1 h in order to analyze the frequency of SCE (SCE2 ). SCE2 analysis was not carried out in the jaundiced and non-jaundiced control groups. 2.4. Analysis of sister chromatid exchange For SCE analysis, 0.5 mL of heparinized blood was drawn from each individual. Cultures were established by adding 0.25 mL of blood to 5 mL karyotyping medium (Biological Industries, Beit Haemek, Israel) with 2% phytohaemagglutinin M (PHA) (Biological Industries, Beit Haemek, Israel), and incubating for 24 h at 37 ◦ C. A 5-bromo-2 -deoxyuridine (BrdU) (Sigma, St. Louis, MO, USA) solution was added to a final concentration of 5 g/mL. Lymphocytes were cultured in the dark for 48 h and metaphases were blocked during the last 2 h with Colcemid (Biological Industries, Beit Haemek, Israel) at a final concentration of 0.1 g/mL. Further processing included hypotonic treatment, fixation, slide preparation and Fluorescein plus Giemsa (FPG) staining for the detection of SCE [15]. Fifty second-division metaphases were scored on coded slides by a single observer, and expressed as the number of SCEs/cell per subject. Staffs performing the SCE analysis were blinded to the study. 2.5. Statistics SPPS 13.0 for Windows® (SPSS Inc., Chicago, IL) was used for statistics in the study. A p-value of <0.05 was considered as statistically significant. The convenience to normal distribution in continuous variables was analyzed by Kolmogorov–Smirnov’s Test of One Sample at the end of which the results were negative, and analysis was continued with non-parametric tests. Results are expressed as means in descriptive statistics with regard to the normal distributed variables. The Kruskal–Wallis and Mann–Whitney U-tests were used to determine significance. Pearson’s Correlation Analysis was used for the assessment of relation between the parametric variables. A Wilcoxon’s Rank Test was performed for the assessment of any difference between the first and second values of the dependent groups.
3. Results Eighty-three newborns were involved in the study, 19 of whom were in the intensive group, 23 in the conventional, 21 in the jaundiced control group, and 20 in the non-jaundiced controls. The groups analyzed did not show statistically significant differences in terms of birth weight, age and weight at the moment of application, route of delivery, and type of feeding. TSB levels differed significantly as expected. No difference was seen between intensive and conventional phototherapy groups (p = 0.4); the TSB levels of these groups were higher than those of the two control
A. Karadag et al. / Mutation Research 676 (2009) 17–20
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Table 1 Baseline demographic data of newborns in the study.
Male/female Gestational age (week) (mean ± SD) Birth weight (g) (mean ± SD) Vaginal/cesarean delivery (%) Admission weight (g) (mean ± SD) Admission age (h) (mean ± SD) Admission TSB level (mg/dL) (mean ± SD)
Intensive phototherapy, n = 19
Conventional phototherapy, n = 23
Jaundiced control, n = 21
Non-jaundiced control, n = 20
pa
11/8 37.8 ± 1.2 3288 ± 493 26.3/73.7 3133 ± 525 111.5 ± 28.1 20.2 ± 1.3
11/12 38.6 ± 1.8 3236 ± 522 35/65 3082 ± 512 114.1 ± 27.7 19.6 ± 1.5
12/9 38.4 ± 1.5 3317 ± 373 26.1/73.9 3168 ± 401 96.2 ± 18.3 13.7 ± 1.5
9/11 38.4 ± 1.8 3219 ± 399 14.3/85.7 3219 ± 399 101.8 ± 25.1 3.6 ± 0.8
0.133 0.23 0.83 0.78 0.424 0.65 0.001b
TSB: total serum bilirubin. a p-Value resembles all groups. b There is no difference between phototherapy groups, and TSB levels are higher than in the two control groups. Jaundiced control group has significantly higher TSB levels than non-jaundiced control group.
groups (p = 0.001). Also, TSB levels of the jaundiced control group were significantly higher than those of the non-jaundiced control group (p = 0.001). Baseline characteristics of the participants were summarized in Table 1. No difference was found between the two phototherapy groups in terms of side effects possibly related to the phototherapy, like weight loss, alterations in body temperature, dermal reactions and daily number of defecations. There was no significant difference between the two phototherapy groups in terms of hemoglobin, hematocrit and TSB levels at the time of application and discharge. However, a significant difference was recorded with regards to phototherapy duration and rate of decrease in bilirubin levels (Table 2), the average duration of treatment in the intensive group being shorter (29.9 ± 11.3 h vs 46.0 ± 14.1 h; p = 0.001), and the rate of decrease in bilirubin per hour being greater (0.26 ± 0.07 mg/(dL h) vs 0.14 ± 0.04 mg/(dL h); p = 0.001). When we take notice of SCE1 , the intensive, conventional and jaundiced controls did not show any difference, whereas these three groups showed an evident increase in SCE compared with the non-jaundiced group (p = 0.001) (Table 3). SCE2 frequency was slightly higher in the intensive group compared with the conventional group, but this was not significant (p = 0.39). SCE1 frequencies were significantly lower than SCE2 frequencies in both phototherapy groups (p = 0.001, p = 0.001, respectively). Neither group had chromosome fragmentation. Correlation analysis made on the variables affecting the SCE1 frequency showed a strong correlation between TSB levels on admission and SCE1 frequency (p = 0.001; r = 0.87) (Fig. 1).
Table 2 Comparison of phototherapy groups. Intensive phototherapy, n = 19 Admission TSB (mg/dL) Discharge TSB (mg/dL) Hemoglobin (g/dL) Hematocrit (%) Phototherapy duration (h) TSB lowering rate (mg/(dL h))
20.2 12.9 17.6 50.8 29.9 0.26
± ± ± ± ± ±
1.3 1.6 1.7 5.6 11.3 0.07
Conventional phototherapy, n = 23 19.6 13.2 17.1 51.6 46.0 0.14
± ± ± ± ± ±
1.5 1.3 1.5 5.1 14.1 0.04
p
0.09 0.58 0.47 0.72 0.001 0.001
TSB: total serum bilirubin.
Fig. 1. The correlation of SCE1 frequencies and admission TSB levels.
4. Discussion We found that both the intensive and the conventional therapies induce an increase in SCE frequencies of T-lymphocytes of newborns during the period of treatment. Moreover, TSB levels >10 mg/dL increased SCE frequencies as well. In fact, phototherapy and bilirubin as a photosynthesizing agent have been known for a long time to have some harmful effects on DNA [4–6,16]. Christensen et al. showed that less DNA damage and cytotoxicity were found in cell cultures after irradiation with green light than after blue light [17]. Wu et al. exposed G0 lymphocytes isolated from newborns to blue or green phototherapy light and found that both colours, but especially blue light, induced SCE [18]. SCE frequency is a more sensitive biomarker of chromosomal changes because of its sensitivity to low levels of genotoxic agents [15,19]. We, too, used the SCE method in this study in order to assess chromosomal change. The first clinical study that showed phototherapy to increase SCE frequency was published by GoyanesVillaescusa et al. who compared SCE frequencies in T-lymphocytes of newborns in three groups of phototherapy, non-phototherapy
Table 3 SCE frequencies in the groups during the study.
SCE1 SCE2
Intensive phototherapy n = 19
Conventional phototherapy n = 23
Jaundiced control n = 21
Non-jaundiced control n = 20
p
9.37 13.5
9.74 13.55
9.23 –
6.17 –
0.001a 0.39
SCE: sister chromatid exchange. a p = 0.001: versus non-jaundiced control.
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but jaundiced, and non-jaundiced [20]. Frequencies were significantly higher in newborns of phototherapy group than in controls. Later, Schwartz et al. reported no difference in terms of SCE frequencies between newborns of phototherapy and control groups [21]. Another study, too, found a similar result [22]. These three studies had fewer participants than ours. Furthermore, gestational ages and application ages were not homogenous, phototherapy starting and cessation indications were not standardized, and, probably most important, only conventional therapy was used in these studies. For that reason our study seems to be the first clinical study investigating chromosomal side effects of intensive phototherapy. We had also shown, by means of the comet assay in a previous study, that conventional phototherapy may have harmful effects on DNA [5], but since only conventional therapy was used we could not conclude whether intensive therapy has chromosomal side effects. Also few data are present in the literature about extrachromosomal effects of intensive therapy. LED phototherapy is reported to increase trans-epidermal water loss to a similar extent as conventional phototherapy [23] and to cause apoptosis in rat intestine much more than does conventional therapy [24]. Yet we could not find any general side effects probably related to the phototherapy in our study. It has been known for a long time that stray light has a decreasing effect on bilirubin levels in neonatal jaundice [25]. In addition, we know that this happens due to the photosynthesizing nature of the specific wavelength on bilirubin [17]. We paid attention to this aspect by shielding the specimens in order to prevent our results from being affected by the stray light. But still we were unable to strictly standardize the conditions since technical problems necessarily emerged, due to the lights in the room or the day–light the patients were exposed to, which could have an influence on the decrease of the bilirubin and SCE levels. However, we ignored this minor effect in the study. One of the additional results of our study was the comparison of the effectiveness of the two therapies, in that the intensive therapy was found more effective in the treatment of jaundice of the newborn. The rate of bilirubin decreased per hour was apparently higher in the intensive therapy group, and the hospitalization duration was shorter. The latter was in concordance with the literature [10,26]. The two most important factors to affect this decrease arise from the overt difference in irradiance between the methods, chosen as approximately 35 W/(cm2 nm) in intensive therapy, and 10 W/(cm2 nm) in the conventional phototherapy. A difference in SCE frequencies was suspected in our study because of this apparent distinction, but the results were nearly the same. We considered the shorter therapy period in the intensive therapy probably accounting for this similarity, despite the stronger irradiance. Although this study was not a longitudinal investigation of longterm effects of phototherapy, its results suggest that the kind of phototherapy widely used in neonatology units can increase chromosomal effects in newborns. In this study we demonstrated that the LED technology and the conventional phototherapy caused a similar increase in SCE frequency in newborns, but no clear chromosomal fragmentations in any of the patients were seen. Higher SCE frequencies were seen in newborns whose TSB was >10 mg/dL than in those with >5 mg/dL, as a result of which bilirubin, independently, was supposed to be a possibly toxic agent to chromosomes. Notwithstanding these data and those in the literature supporting chromosomal side effects of phototherapy, none is serious enough to constitute a drawback for its usage in newborn’s jaundice. There is no evidence that the chromosomal side effect of the phototherapy is permanent and that it causes chronic diseases or cancer in further years. For that reason, wider, randomized and controlled clinical studies investigating the relation between phototherapy and chromosomal side effects are needed. Given the incomplete understanding of these problems, the safest approach
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