E L S E V I E R Mutation Research 334 (1995) 235-241 Environmental Mutagenesis

Effect of renutrition on the proliferation kinetics of PHA stimulated lymphocytes from malnourished children

R o c i o O r t i z a,*, C a r o l i n a C a m p o s a, J o s 6 L. G 6 m e z a, M i g u e l E s p i n o z a b,

M i g u e l R a m o s - M o t i l l a c, M i g u e l B e t a n c o u r t a

a Departamento de Ciencias de la Salud, Universidad Autdnoma Metropolitana-Iztapalapa, C.P. 09340 M~xico D.F., Mexico b Hospital Infantil de Iztacalco, Secretar{a de Salud del D.D.F., C.P. 08500, M~ico D.F., Mexico

e Centro de Rehabilitacidn Nutricional Cruz Blanca A.C., C.P. 04040, Coyoacdn M~xico D.F., Mexico

Received 11 March 1994; revision received 12 August 1994; accepted 31 August 1994

Abstract

The fraction of lymphocytes that responded to phytohemagglutinin (PHA) stimulation and initiated cellular proliferation (stimulation index or SI) was determined in groups of healthy and severely malnourished children. SI was determined again in the latter group after a period of nutritional recovery. The proportion of interphasic cells showing PHA response was assessed adding bromodeoxyuridine to the culture, so proliferative nuclei appear big and stain light blue, with dispersed granular chromatin and apparent nucleoli, while non-proliferative nuclei look small, stain red, and have compact and homogeneous chromatin. In mitotic nuclei, differential staining of sister chromatids made it possible to distinguish cells that had gone through one, two and three or more proliferation cycles. Based on the data obtained from interphase nuclei and mitosis, the SI was estimated at 48 and 72 h of culture. SI were higher in lymphocytes from healthy children than in those from children with severe malnutrition, even after the period of nutritional recovery. However, the SI was significantly higher in lymphocytes from malnourished children after nutritional recovery. Although in these children more cells are stimulated, there seems to be still damage that causes a cycling delay.

Keywords: Protein-calorie malnutrition; Renutrition; Human lymphocytes; Stimulation index; Average generation time

I. Introduct ion

Severe protein-calorie malnutrit ion (PCM) is a serious public health problem in Mexico as well as in other countries. The bet ter understanding of the biochemical pathology of the disease, and

* Corresponding author. Fax (5) 724-47-27.

the introduction of more successful t reatments for severe infections have markedly decreased PCM associated mortality. The increased survival frequency of PCM patients has caused concern regarding the possible long-term consequences of this illness. It is therefore important to evaluate possible persistent physiological effects resulting from PCM which may represent a potential haz- ard to the health of these patients (Betancourt et al., 1989).

0165-1161/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0165-1161(94)00082-4

236 R. Ortiz et al. /Mutation Research 334 (1995) 235-241

Alterations in cellular proliferation kinetics have been observed in phytohemagglutinin (PHA) stimulated lymphocytes from PCM children, as measured by the bromodeoxyuridine (BrdU) de- pendent sister-chromatid differential staining technique. Murthy et al. (1982) reported that PCM lymphocytes showed slower proliferation rates, while other authors (Mutchinick et al., 1979; Gonzalez et al., 1990; Ortiz et al., 1994) reported faster proliferation rates in malnourished chil- dren.

Although these results demonstrate that there is an altered in vitro proliferative rate for stimu- lated lymphocytes, they do not provide informa- tion on the proportion of lymphocytes that have actually responded to the mitogen. Nevertheless, it is possible to score the fraction of proliferative and non-proliferative lymphocytes.

The stimulation index (SI) represents the frac- tion of mitogen responsive lymphocytes in cul- ture, which is estimated by detecting proliferating interphase cells according to the morphology, color and size of the nuclei, and detecting the proportion of mitotic cells undergoing the first, second, and third or subsequent proliferative cy- cles (Pincu et al., 1986).

The aim of this study was to compare the SI of lymphocytes from PCM children with that of lym-

phocytes from normal children, and to assess this index after a period of nutritional recovery.

2. Materials and methods

Six severely malnourished children (five with marasmus and one with kwashiorkor) were stud- ied during the severe phase of their illness, and after a nutritional recovery period of 2-6 months. When the study began, all malnourished children had received antibiotic therapy for infections (di- cloxacillin or amikacin for 7-10 days) 2 weeks prior to sampling.

The characteristics of these children are shown in Table 1. Marasmic children had a 35-48% body weight/height deficit, according to tables for Mexican children (Ramos-Galvfin, 1976). Their ages ranged from 6 to 13 months, four were boys and two were girls. The kwashiorkor child was an 8 month old boy with an 8% deficit. After a period of medically supervised renutrition, the body weight/height deficit of the marasmus chil- dren improved in all cases (Table 1). The clinical features proved that all children were clearly under recovery.

The control group consisted of three girls and three boys who were well-nourished, aged 18-33

Table 1 Characteristics of the well-nourished children and of the malnourished children before and after nutritional recovery

Child Sex Age Height Weight Deficit Recovery Height Weight Deficit (months) (cm) (kg) (%) period (cm) (kg) (%)

(months)

Well-nourished GSS M 18 80 11.0 CASH F 19 82 11.5 ELA M 24 83 12.0 SGN M 32 94 16.3 RMC F 33 90 15.1 A C R F 33 93 15.5

Malnourished + R A M M 6 62 4.6 * IAO M 8 61 6.8 + NAY F 9 62 4.2 + TTR F 9 56 3.5 + BCR M 7 62 3.9 + FJV M 13 65 5.4

35.0 5 68 7.5 15.4 8.3 3 67 8.2 6.2

44.6 2 73 7.6 17.5 48.4 5 66 6.8 22.0 46.7 6 69 8.6 6.0 37.9 4 69 8.3 13.0

+ , Marasmus; *, kwashiorkor.

R. Ortiz et al. /Mutation Research 334 (1995) 235-241 237

months and had normal weight and height for their age (Table 1).

Peripheral blood lymphocytes were cultured in the dark for 48 and 72 h at 37°C in 5 ml McCoy 5A medium (Microlab, Mexico), supplemented with 0.2 ml PHA (Microlab) and 2.5 X 10 -5 M of BrdU (Sigma, USA). 10 /zg of colcemid (Micro- lab) was added 1 h before harvesting. Cells were treated with hypotonic solution (KC1 0.075 M) and fixed with methanol-acetic acid 3 : 1.

Several drops of cellular suspension were placed on a cold slide and quickly flame-dried. Differential staining of sister chromatids was done with the fluorescence plus Giemsa method (Perry and Wolff, 1974). Cells were stained with 33258 Hoechst (9 tzg/ml) (Sigma) for 30 min, mounted in S6rensen buffer, pH 6.8, exposed to black light for 3.5 h and stained in 5% Giemsa for 5 min.

At each harvest time, 1000 interphase nuclei were analyzed and classified as proliferative or non-proliferative cells according to their color, size and appearance. Non-proliferative cells have not synthesized DNA in culture, so they have not incorporated BrdU when harvested. The nuclei of these ceils are small with compact chromatin and show homogeneous red staining.

Proliferative cells have synthesized DNA at least once in culture, or have gone through one or more cycles of DNA synthesis and have there- fore incorporated BrdU. The nuclei of these cells are large, show red speckles on a bluish back-

ground and have apparent nucleoli (Pincu et al., 1984; Ostrosky et al., 1987).

In the same cultures, 100 consecutive metaphases were evaluated to determine each one as undergoing either the first, second, and third or subsequent replication cycle according to their differential staining patterns (Tice et al., 1976). According to the metaphase data, the av- erage generation time (AGT) (Ivett and Tice, 1982) was calculated as follows:

AGT (h) = time (in hours) since onset BrdU ex- posure/replication index (RI)

RI = l(number of first cycle mitosis) + 2(number of second cycle mitosis)+ 3(number of third or more cycle mitosis)/number of analyzed mitosis (Schneider and Lewis, 1981).

The SI was estimated according to the formula described by Pincu et al. (1986) with modifica- tion, as follows:

SI = [Bo/(Bo + non-proliferative nuclei)] × 100

Bo = Proliferative nuclei/RI

Where Bo represents the number of proliferating interphase cells, RI is the replication index and SI is the fraction of cells that are stimulated to go into DNA synthesis.

The data were compared using the non-para- metric Mann-Whitney test. The SI of lympho- cytes from malnourished children before and af-

Table 2 Proliferative and non-proliferative cells, percentage of metaphases in first, second or third replication cycle and replication index at 48 and 72 h of culture, in lymphocytes from well-nourished, malnourished and nutritional recovery children

Hours Well-nourished Malnourished Nutritional recovery

Cells Metaphases Cells Metaphases Cells Metaphases

NP P+ 1st 2nd 3rd RI NP P 1st 2nd 3rd RI NP P 1st 2nd 3rd RI

4 8 X 306 694 56 41 3 1.5 * 448 552 30 65 5 1.8 * 450 550 65 32 3 1.4 SD 35 33 21 19 3 0.2 56 56 14 12 4 0.2 51 51 21 20 6 0.2

72"X 210 790 1 14 85 2.9 362 638 9 21 70 2.6 357 643 12 46 42 2.3 SD 79 79 1 4 4 0.1 85 85 7 9 14 0.2 10 10 11 17 20 0.3

N = 6 ; * N = 5 + Statistieallydifferent vs. malnourished and nutritional recovery, P < 0.02. NP, Non-proliferative cells; P, Proliferative cel ls ;RI , replication index.

238 R. Ortiz et al. /Mutation Research 334 (1995) 235-241

Table 3

Average generation time (AGT) in lymphocytes of well-nourished (WN), malnourished (MN) and nutritional recovery (NR) children

48 h 72 h

WN MN NR WN MN NR

13.41 13.71 ND 17.32 17.65 19.28

14.46 12.70 17.78 16.60 17.58 19.92

12.97 ND 15.00 16.60 18.75 25.80

18.90 13.50 14.72 16.55 19.67 19.67 16.78 13.04 20.51 17.02 16.55 23.76

20.17 16.22 21.43 16.96 20.42 18.90

16.11 13.83 17.89 16.89 18.44 21.22 SD 2.99 1.39 3.07 0.31 1.45 2.85

ND, not determined. 48 h

WN vs. MN: U = 6, p < 0.063

WN vs. NR: U = 6, p < 0.063

MN vs. NR: U = 2, p < 0.016

72 h WN vs. MN: U = 1, p < 0.002

WN vs. NR: U = 0, p < 0.001

MN vs. NR: U = 4, p < 0.013

ter the renutrition period were compared using Student's paired t-test.

3. Results

Table 2 shows that the number of proliferative nuclei was significantly higher at all harvest times in cultures from well-nourished children than in the other two groups (p < 0.02).

However, the RI was higher in malnourished children at 48 h of culture (p < 0.02) but in contrast significantly lower than in cells from

well-nourished children at 72 h of culture (p < 0.02). Moreover, the RI was similar in nutritional recovery and well-nourished children at 48 h, but lower in the first group at 72 h. This is related to the higher percentage of second cycle metaphases and the concomitant lower percentage of first cycle metaphases at 48 h in lymphocytes of mal- nourished children. At 72 h of culture, the per- centage of third and more cycle metaphases was higher in well-nourished children. Besides, at this time first cycle metaphases were still present in the other two groups of children (Table 2).

The AGT estimation (Table 3) at 48 h was

Table 4 Stimulation index (%) in lymphocytes from well-nourished (WN), malnourished (MN) and nutritional recovery (NR) children

48 h 72 h

WN MN NR WN MN NR

60.65 48.56 ND 51.38 33.52 43.53

59.88 ND 49.65

62.37 36.24 41.76 63.46 41.08 45.58

68.25 48.47 56.23

61.00 41.57 47.39 SD 5.56 6.89 5.79

45.48 50.23 48.13 47.20 36.32 31.01

64.49 42.25 58.51

49.40 28.30 40.15 70.16 37.59 34.36

70.65 50.54 56.99

57.89 40.87 44.86

11.81 8.62 11.56

ND, not determined. WN vs. MN 48 h: U = 0, p < 0.002 72 h: U = 3, p < 0.008

WN vs. NR U = 0, p < 0.002

U = 5.5, p < 0.026

MN vs. NR t = 5.7, p < 0.02 t = 1.1, p > 0.05

R. Ortiz et al. / Mutation Research 334 (1995) 235-241 239

lower in malnourished cells than in well-nourished cells (X = 13.8 + 1.4 and X = 16.1 + 3.0 respec- tively), this difference was statistically marginal (p < 0.06). In contrast, at 72 h of culture the A G T estimation was higher in malnourished than in well-nourished children (X = 18.4 + 1.5 and X = 16.9 + 0.3 respectively; p < 0.002). AGT was higher in nutritional recovery cells than in mal- nourished ceils at 48 h (X = 17.9 + 3.1; p < 0.016) and 72 h (X = 21.2 + 2.9; p < 0.013), and was also higher than in well-nourished cells at 48 and 72 h harvesting times (p < 0.06 and p < 0.001).

Based on the nucleus and metaphase data, SI values were estimated in all lymphocyte cultures from well-nourished, malnourished and renutri- tion children as shown in Table 4.

Cells from the control group showed a higher PHA stimulation response as compared to the other study groups. The rate of proliferating cells (SI) at 48 and 72 h was significantly higher in the control group (61.0% and 57.9%) as compared to malnourished children (41.6% and 40.9%): U = 0, p < 0.002; U = 3, p < 0.008 for 48 and 72 h of culture respectively. SI was also significantly higher in the control group than in the children in the nutritional recovery period (47.4% and 44.9%): U = 0, p < 0.002; U = 5.5, p < 0.026 for 48 and 72 h of culture respectively.

The group of children under renutrition had higher SI estimations than the malnourished chil- dren, but not as high as the estimations obtained from the group of well-nourished children. The paired t-test was used to compare the former two groups, and a statistically significant difference was obtained at 48 h of culture (t = 5.7; df = 3; p < 0.02). However, at 72 h of culture, the SI increased only in three of the six nutritional re- covery children, did not change in two more, and decreased in one. The differences were not statis- tically significant.

4. Discussion

As revealed by the SI estimated at 48 and 72 h of culture, lymphocytes of well-nourished chil- dren showed a higher rate of stimulation as com- pared to lymphocytes from the other two groups,

and lymphocytes of malnourished children showed an impaired PHA stimulation response. Although the malnourished children showed a lower fraction of cells in active proliferation, their cultures had shorter A G T at 48 h than the other two groups. This may be a consequence of the responsive lymphocytes from malnourished chil- dren having a shorter lag period between PHA exposure and transition from the GO to the G1 phase in the cell cycle, which may occur when the 'starving cells' from malnourished children are placed in a rich culture medium. However, these cells did not seem to have shorter cell cycle times, because at 72 h of culture the A G T was higher in cells from malnourished children.

In a previous study we had analyzed RI in lymphocytes exclusively at 48 h of culture, and we suggested that the higher RI observed in mal- nourished children could be due to an increased PHA response a n d / o r a shorter cell cycle subse- quent to stimulation (Ortiz et al., 1994). The results presented here support only the first alter- native.

The lower SI observed in malnourished chil- dren could be related to a decreased production of several cytokines, as well as a decreased pro- portion of helper a n d / o r inducer T lymphocytes, or to a deficiency of essential factors in the plasma of these patients for the normal development and function of thymus derived lymphocytes (Chandra, 1991; Hoffman-Goetz et al., 1984; Beatty and Dowle, 1978).

We previously observed a severe delay in cell proliferation and a lower rate of cells in active proliferation in bone marrow cells from severely malnourished rats (Betancourt et al., 1992). The results of the present study also show that lym- phocytes from malnourished children have a lower rate of actively proliferating cells, with a signifi- cant delay in the duration of the generation cycle, evident at 72 h of culture.

Although the proliferation of lymphocytes from malnourished children may have been affected by the previous exposure to antibiotics, this therapy had concluded at least 2 weeks before the chil- dren were sampled in all cases. Further studies will be necessary to asses the effect of these drugs on cells from malnourished children.

240 R. Ortiz et al. /Mutation Research 334 (1995) 235-241

Although the children showed clear clinical signs of recovery after renutrition, they still had some degree of weight/height deficit. At the cellular level, the SI estimated in lymphocytes from this group generally increased after recov- ery, but remained lower than in the cultures from well-nourished children. It is important to point out that although a greater number of cells were stimulated, the higher AGT means that the cell cycle impairment persists in malnourished chil- dren even after a period of nutritional recovery.

The results presented here indicate that renu- trition allows T lymphocytes to partially improve their ability to respond to a specific mitogen (PHA). This is in agreement with previous studies (Gorodezky et al., 1986) reporting that lympho- cytes from children under renutrition increased DNA and RNA synthesis in response to PHA stimulation.

In conclusion, the proportion of cells that re- spond to PHA stimulation is lower in lymphocyte cultures of malnourished children, but the cells that show a response do it in a more efficient manner apparently by reducing the lag period before entering the cell cycle. However, there is a delay in the cell cycle in lymphocytes of malnour- ished children.

The fraction of lymphocytes from recovering children that responded in culture was higher than that observed in malnourished children, but did not reach SI estimations observed in well- nourished children. This partial improvement could be related to the recovery of the T lympho- cyte mediated immune cellular response. Al- though more cells are stimulated, there seems to be still some damage that causes a cycling delay and which is not related with the stimulation by PHA.

Acknowledgements

We wish to thank Dr. Raymond Tice for his suggestions and helpful discussion, Edith Cort6s for her technical assistance and Biol. Cristina Gonzfilez for reviewing the manuscript. Sup- ported partially by Grant 400262-5-1485M from

the National Council of Science and Technology (CONACyT, Mexico).

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