Br. J. Anaesth.
(1986), 58, 540-543
EFFECT OF HALOTHANE AND NITROUS OXIDE
ANAESTHESIA ON NATURAL KILLER LYMPHOCYTES
FROM PATIENTS WITH BENIGN AND MALIGNANT
BREAST DISEASE
C. D. M. GRIFFITH AND M. B. KAMATH
Natural killer (NK) cells are a lymphocyte subset
which can recognize and kill tumour cells without
prior processing of tumour cell specific antigen.
Hence, they are widely believed to defend against
the development of primary tumours, and the
metastatic spread of established tumours in the
host (Herberman and Ortaldo, 1981). This
phenomenon is known as "natural cytotoxicity."
Many facets of the host immunological
response are known to be depressed following
anaesthesia and surgery (Walton, 1979) and this
could possibly be implicated in the dissemination
of tumour cells following surgery for malignant
disease.
The aim of the present study was to determine
the effects of halothane and nitrous oxide
anaesthesia on the capacity of human natural killer
lymphocytes to kill the tumour cell line K562.
PATIENTS AND METHODS
Patients
Twenty female patients undergoing surgery for
benign and malignant breast disease were studied
after giving informed consent. Ten patients (mean
age 43 yr, range 35-60 yr) underwent excision
biopsy of breast for benign breast disease and 10
patients (mean age 54 yr, range 36-73 yr) under-
went segmental or total mastectomy for breast
cancer. Whole blood was taken from an antecubital
vein before surgery, heparinized and stored at
4 °C overnight before study.
C . D . M . GRIFFITH, M.D., F.R.C.S.(ED.); M. B. KAMATH*,
M.B.B.S., F.F.A.R.C.S.; Professorial Surgical Unit, City Hospital
and University Department of Anaesthesia, University
Hospital, Nottingham.
* Address for correspondence: Academic Department of
Anaesthesia, C Floor, East Block, University Hospital, Clifton
Boulevard, Nottingham NG7 2UH.
SUMMARY
The effect of halothane and nitrous oxide on the
capacity of natural killer (NK) lymphocytes from
female patients with benign and malignant breast
disease to kill the tumour cell line K562. was
studied in vitro. There was no depression of
activity of NK lymphocytes when exposed to 2 %
halothane and 66% nitrous oxide either alone or
in combination. However, NK lymphocyte activity
was depressed at higher concentrations of
halothane and the decrease in activity was
significant (P < 0.01) when 4% halothane was
used. These findings suggest that exposure to
clinically-used concentrations of halothane and
nitrous oxide does not inten'ere with the NK
lymphocyte response of the host.
Anaesthesia
Equal volumes of whole blood and RPMI 1640
culture medium with 10% fetal calf serum (Flow
Laboratories, Irvine, Scotland) were mixed. One
hundred microlitre of this mixture was placed
into round-bottomed micro test plates (Falcon
Products, Becton Dickinson, Ca., USA). The test
plates were then placed in sealed 1-litre chambers
and exposed to air (control) or flushed with an
anaesthetic mixture delivered from a Boyle
machine at a fresh gas flow of 9 litre min"1 for
5 min of either 2 % halothane + 98 % oxygen, or
66 % nitrous oxide + 34 % oxygen or 2 % halothane
plus 66 % nitrous oxide in oxygen. Each chamber
was incubated at 37 °C for 2 h before assessment
of cytotoxicity.
In a further part of the study, heparinized whole
blood from five patients (two patients suffering
from benign disease and three from breast cancer,
NK CELLS, HALOTHANE AND NITROUS OXIDE 541
mean age 42 yr, range 34-53 yr) was diluted with % chromium 51 release =
RPMI 1640 (+10% FCS) and exposed to 2 x (half SN)
increasing concentrations of halothane (2, 3 and xlOO
(Cells + half SN) + (half SN)
4 %) in oxygen for 2 h at 37 °C before assessment
of cytotoxicity. where SN = supernatant.
Percentage cytotoxicity was then calcutated as
follows:
Assay of cytotoxicity % cytotoxicity =
The whole blood natural cytotoxicity assay test release —spontaneous release xlOO
(Rees and Platts, 1983) was used to test NK cell 100 —spontaneous release
function rather than using separated lymphocytes Spontaneous release represented the chromium 51
since it is more representative of the in vivo released from K562 target cells incubated in
situation. culture medium alone.
The leukaemic cell line K562 was used as the
target cell. Cells were labelled with chromium Statistical analysis
51-labelled Na^CrO^ 100 uCi (Radiochemical Paired and unpaired Student t tests were used,
Centre, Amersham, Bucks), washed in RPMI as appropriate.
(10% FCS) and resuspended at 106 cells ml~l.
One hundred microlitre of labelled K562 cells RESULTS
was added to 0.1-ml volumes of whole blood (half
dilution) after exposure to either air (control) or There was no significant difference between the
the anaesthetic agent under study. Each test was mean percentage cytotoxicity of the 10 patients
performed in triplicate. with benign breast disease and the 10 patients with
carcinoma of the breast (table I).
The test wells were incubated at 37 °C in a 5 %
carbon dioxide-95 % air atmosphere for 6 h. Then TABLE I. Percentage cytotoxicity of whole blood (one-half
0.1 ml of supernatant was removed into separate dilution) to K562 exposed to air (control). Differences between
wells. The test wells were dried in an oven, groups not significant (unpaired t test)
sprayed with Nobecutane (Astra Pharmaceuticals,
Benign cancer
Kings Langley, Herts) and individual wells Benign group group
counted in a Wilj gamma spectrophotometer. The (n = 10) (n = 10)
release of chromium 51 is directly proportional to
tumour target cell killing and was calculated as Cytotoxicity (%) 16.7 16.9
follows: SD 7.3 5.9

401- 4Ot- 40|-

30 30 30-

~ 20 20 20
3
10 J . 10

Air 2ZHal Air 67ZN2O Air 2ZHal+

Mean 16.7 15.5 W.7 19.2 16.7 16.6

cytotoxicity
4 — ns <
Paired (test
ISO 7.3 7.1 7.3 7.2 7.3 6.1

FIG. 1. Percent cytotoxicity of whole blood (one-half dilution) to K562 cells after exposure to halothane
and nitrous oxide (patients with benign breast disease).
542 BRITISH JOURNAL OF ANAESTHESIA
40 401- 40r-

30 30 30

% 20 20 20

s
o
10 10

Air 27. Hal Air 672 N2O Air 2lHat*

66lN20
Mean
cytotoxicity 16.9 179 16.9 17.5 16.9 16.9
Paired nest 4 — ns — »
iso 5.9 7.1 5.9 7.6 5.9 8.9

FIG. 2. Percent cytotoxicity of whole blood (one-half dilution) to K562 cells after exposure to halothane
and nitrous oxide (patients with breast cancer).

In the group of patients with benign breast

disease (fig. 1), exposure of their blood to 2%
halothane + 98 % oxygen, 66% nitrous oxide +
34% oxygen or 2% halothane, 66% nitrous
oxide + 32 %, oxygen did not alter NK cell cyto- 20

toxicity to K562 tumour cells when compared with

cytotoxicity of blood exposed to air alone.
Similarly, in the patients with breast cancer
(fig. 2), NK cytotoxicity of whole blood to K562 g
tumour cells was not significantly different when
incubated in 2% halothane+ 98% oxygen, 66%
I
nitrous oxide+ 34% oxygen or 2% halothane,
66 % nitrous oxide + 32 % oxygen or air alone.
However, NK cell cytotoxicity from the addi- Air 2lHa\ 32 Hal 4ZHal
tional five patients was depressed (fig. 3) after Mean
14.9 114 as
exposure to 3 % halothane + 97 % oxygen for 2 h, cytotoxicity
*SD 11.2 8.7 7.4 6.3
and this depression became statistically significant
Paired t test nt na P <0.01
(P < 0.01) when 4% halothane + 96 % oxygen was
used. This appears to demonstrate a dose FIG. 3. Cytotoxicity of whole blood (one-half dilution) to K562
dependent inhibition of natural killer lymphocyte cells after exposure to increasing concentrations of halothane
cytotoxicity after exposure to increasing concen- (five patients).
trations of halothane.
and Soothill, 1974) and there was also a reduction
in circulating T lymphocytes and inhibition of the
DISCUSSION
mixed lymphocyte culture reaction (Slade et al.,
Depression of the lymphocyte-mediated immune 1975).
response has been reported following surgical Halothane, in vitro, inhibits lymphocyte trans-
operations performed under general anaesthesia. formations (Cullen, Sample and Chretien, 1972),
Studies have concluded that the depression of cell division (Nunn, Lovis and Kimball, 1971) and
lymphocyte function (Riddle, 1967) was related to motility (Nunn, Sharp and Kimball, 1970), and
the degree of surgical stress (Cullen and Van Belle, both halothane and nitrous oxide inhibit antibody
1975) and that this was more important than dependent cellular cytotoxicity (Cullen, Duncan
exposure to anaesthetic agents. However, lympho- and Ray-Keil, 1976) mediated by K lymphocytes.
cyte transformation to mitogens was reduced The NK lymphocyte, because of its unique
following induction of anaesthesia (Espanol, Todd ability to recognize and kill tumour cells, without
NK CELLS, HALOTHANE AND NITROUS OXIDE
processing tumour specific antigen, is thought to
form a primary immune defence mechanism
against the development of tumours and has a
secondary role in defence against tumour meta-
stases. Natural killer lymphocyte cytotoxicity in
patients with breast cancer was found to be
depressed 3 days after mastectomy (Uchida, Kolb
and Micksche, 1982); however, cytotoxicity was
not assayed following the induction of anaesthesia
or during the surgical procedure. A further study
showed that the induction of anaesthesia with
inhalation anaesthetic agents (nitrous oxide and
halothane) or i.v. anaesthetic agents (etomidate
and fentanyl) did not affect the NK activity of
patients with benign and malignant abdominal
conditions. NK activity was increased during the
surgical procedure and for 48 h following operation
as the result of an increase in circulating NK
lymphocytes (Griffith et al., 1984).
In this study we have shown that exposure to
clinically used concentrations of nitrous oxide and
halothane either alone, or in combination, had no
effect on the ability of NK lymphocytes to kill the
tumour cell line K562. This investigation was
designed to simulate the maximum time required
for a mastectomy and it is reassuring that this
particular limb of the immune response was found
to be intact after exposure to clinical concentrations
of the anaesthetics.
When the concentration of halothane was
increased to 3% and 4%, marked inhibition of
natural cytotoxicity to tumour cells was shown
which was significant after exposure to 4%
halothane (P < 0.01, Student's paired t test).
However, it is unlikely that this concentration of
halothane would be used for a prolonged period
in clinical practice.
The mechanism of inhibition of natural killer
lymphocyte activity after exposure to high concen-
tration of halothane is unknown, but the local
anaesthetics lignocaine and procaine are known to
inhibit NK cell activity in a dose-dependent
manner (Takagi et al., 1983) probably by changing
the cell membrane which will then affect recog-
nition and binding of target cells.
Anaesthetic agents are known to cause destruc-
tion or depolymerization of the microtubular
structure of cells in vitro (Hinkley and Telser,
1974). In addition, one of the theories of the
mechanism of general anaesthesia may be its effect
on membrane proteins and on ion transport across
the membrane (Mullins, 1954). This membrane
effect could be the mechanism behind the decrease
543
in NK activity seen after exposure to high
concentrations of halothane in vitro. Th evidence
from this study, however, shows that NK
lymphocyte activity from patients with benign
and malignant breast disease is not adversely
affected by exposure to clinically-used concentra-
tions of the inhalation anaesthetic agents halothane
and nitrous oxide.
ACKNOWLEDGEMENTS
The authors are grateful to Dr R. C. Rees for supplying the
K562 cells and also to Professor R. W. Blarney for his
co-operation in the study.
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