Permeation and toxicity of ethylene glycol and methanol in larvae of Anopheles gambiae
1 Department of Biochemistry and Cellular and Molecular Biology, University
of Tennessee, Knoxville, TN 37932-2575, USA
2 Department of Chemistry, University of Tennessee, Knoxville, TN 37996,
USA
* Author for correspondence at present address: Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Shriners Hospital for Children, 51 Blossom Street, Boston, MA 02114, USA (e-mail: xiang-hong_liu{at}hms.harvard.edu)
Accepted 31 March 2003
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Summary |
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Key words: cryoprotectant, permeation, toxicity, ethylene glycol, methanol, NMR, larvae, mosquito, Anopheles gambiae
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Introduction |
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Cryopreservation of any biological material requires that cryoprotective
agents (CPAs) are present in the cells either to prevent so-called
`solution-effect' injury (Mazur,
1970) during classical slow-freezing procedures or to prevent
intracellular ice formation during vitrification procedures that involve
cooling at high rates. The former approach is used in the cryopreservation of
most mammalian cells; the latter, vitrification, was used by Steponkus et al.
(1990
) and Mazur et al.
(1992a
) in the successful
cryopreservation of Drosophila embryos. Both approaches demand that
the cells in question be permeable to both water and CPAs. Unfortunately,
native Anopheles eggs show poor permeability to water and are
essentially impermeable to ethylene glycol (EG;
Valencia et al., 1996a
), the
CPA used in the cryopreservation of Drosophila
(Mazur et al., 1992a
;
Steponkus et al., 1990
) and
house flies (Wang et al.,
2000
). Native eggs of Drosophila and house flies are also
impermeable to both water and CPA; however, procedures were developed to
successfully permeabilize them and thereby permit cryopreservation. The
permeability barrier in the eggs of these two species appears to be a wax
layer lying on the surface of the endochorion
(=vitelline membrane), the innermost of the two shells that surround the
embryo proper. An essential component of the permeabilization procedure is
exposure to the alkanes hexane or heptane, compounds that presumably
solubilize the wax layer. Unfortunately, these procedures are not applicable
to the Anopheles eggs (Valencia
et al., 1996b) or are applicable to only a limited extent. Early
eggs of Anopheles also appear to have a wax layer. The problem is
that they develop a second permeability barrier between 8 h and 12 h after egg
laying that is refractory to removal by alkanes. This second barrier is
probably related to tyrosine cross-linking in the endochorion of the egg
(Mazur et al., 2001
). To date,
we have found no method to breach this second barrier or to prevent it from
forming without killing the egg.
An alternative to cryopreserving eggs would be to cryopreserve larvae.
Recently, we found (X.-H. Liu, unpublished data) that untreated 1st instar
Anopheles larvae were uniformly stained by rhodamine B, which has a
molecular mass (Mr) of 479, so it is reasonable to predict
that the larvae will probably be permeable to CPAs with low molecular masses,
such as EG (Mr=62) and methanol
(Mr=32). To verify this assumption, we applied nuclear
magnetic resonance (NMR) spectroscopy to measure the concentration of CPAs
within the larvae. NMR is a phenomenon that occurs when certain nuclei (such
as 1H) that possess an intrinsic magnetic property imparted by
their spin are immersed in a powerful static magnetic field and are
simultaneously exposed to a second oscillating magnetic field
(Ault and Dudek, 1976). For
example, when a proton with a spin is placed in an external magnetic field,
the spin vector of the proton aligns itself with the external field, just as a
magnet would. This proton can undergo a transition between the two energy
states by the absorption of a photon and can jump to the less stable
orientation (higher energy state) when the frequency of the oscillating
electromagnetic signal matches the resonance frequency of the proton or the
energy difference between the two states. Each proton in a chemical has a
characteristic resonance frequency, which is affected by the neighboring
electrons since the circulation of the electrons creates small magnetic fields
that can oppose (generally) or enhance the externally applied field. The
variation in resonance frequency of a nuclear spin due to the chemical
environment around the nucleus is referred to as a chemical shift. Removal of
the oscillating magnetic field causes the proton to revert to the more stable
orientation (lower energy state) and emit electromagnetic radiation, which is
the NMR signal. Protons with different chemical environments produce separate
NMR signals at different chemical shifts. The intensity of the NMR signal at a
given frequency is proportional to the number of protons with that
characteristic resonance frequency. In Fourier-transformed spectra, the NMR
signals yield NMR peaks. Calculation of the integrated area of the peaks will
permit determination of the molar ratio of different protons and thereby
measurement of the concentrations of a specific chemical. These principles
have been applied to study the permeation of dimethyl sulfoxide (DMSO;
Me2SO) in rat liver (Fuller and
Busza, 1994
; Fuller et al.,
1989
), rat carotid artery
(Bateson et al., 1994
) and
rabbit and porcine corneas (Taylor and
Busza, 1992
; Walcerz et al.,
1995
; Wusteman et al.,
1999
), ethylene glycol in the rabbit common carotid artery
(Wusteman et al., 1995
) and a
number of CPAs in human ovarian tissue
(Newton et al., 1998
). Proton
NMR has thus been shown to be a very useful method for measurement of the
kinetics of CPA permeation in tissues. The prime objective of the present
study was therefore to use proton NMR to determine the kinetics of permeation
of EG and methanol into larvae of Anopheles mosquitoes. A second
objective was to determine the resulting toxicity of the CPAs as a function of
permeation. While our underlying goal is cryopreservation, the permeation of
solutes in multicellular systems such as larvae is of broader biological
relevance. We believe that this study illustrates the applicability of NMR to
this broader question.
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Materials and methods |
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Determination of the kinetics of permeation of ethylene glycerol and
methanol into 1st instar larvae
The 1st instar larvae were immersed in 1.5 mol l-1 ethylene
glycol (EG; Sigma Chemical Co., St Louis, MO, USA) or 1.5 mol l-1
methanol (GR Anhydrous; EM Science, Gibbstown, NJ, USA), which were prepared
in Millipore water, for different period of times (1, 15, 30, 60, 120, 180,
240, 360 and 480 min) in a 15 ml centrifuge tube (Corning Inc., Acton, MA,
USA) at room temperature (2224°C). After the appropriate exposure
time, approximately 100200 larvae were pipetted to a nylon cell
strainer (70 µm, Becton Dickinson) to drain the CPA solutions. The strainer
was then blotted with facial tissue and the larvae were briefly washed using 1
ml H2O to remove the residual surface CPAs.
NMR measurements require that the larvae are in deuterium oxide
(D2O) rather than water to eliminate proton signals from the
solvent. The H2O on the larval surface was removed by washing them
twice with 99.90% D2O (Aldrich Chemical Co., Milwaukee, WI, USA).
The strainer was briefly blotted following each washing. The whole washing
procedure took less than 2 min, so the potential washout of permeated CPA was
minimal. The larvae were then suspended in 0.2 ml of high-purity
D2O (99.96%; Aldrich Chemical Co.) and immediately transferred
to a 5 mm symmetrical D2O matched NMR microtube (Catalog no.
BMS-005V; Shigemi, Inc., Allison Park, PA, USA) for measurements. Once the
larvae were in the NMR tubes, efflux of CPA from the larvae into the
surrounding medium would be of no significance. NMR measurements were carried
out using the same NMR parameters as those used for the standard solutions
described below. Each time point was repeated three times for both CPAs at
each exposure time to the CPA.
For experiments involving determination of the kinetics of CPA efflux (washout), larvae that had previously been exposed to 1.5 mol l-1 EG for 4 h or to methanol for 1 h were put in a nylon cell strainer to briefly drain the CPA solution and lightly blotted. The strainer with the larvae was then transferred to a Falcon tissue culture dish (35 mmx10 mm; Becton Dickinson) containing 5 ml Millipore water. After sitting in the water for 15 min, 30 min, 60 min, 120 min (only for EG) or 240 min (only for EG), approximately 100 larvae were removed and placed in another strainer and washed twice with D2O to remove surface water. The subsequent procedures for the NMR measurements were the same as described above.
NMR measurements on standard solutions of EG and methanol
In order to determine the concentration of EG and methanol in the larvae
versus exposure time, it was necessary to first determine the NMR
proton signals generated by a series of solutions of EG and methanol at a
range of concentrations. These standards (010 mol l-1) were
prepared in Millipore water. Specifically, from the NMR peaks we determined
the molar ratio of the NMR peaks attributable to the protons in the
CH2- groups of EG and the CH3- group of methanol to the
area of the peak due to -OH groups. These peaks occur at different chemical
shifts. Concentrations can be calculated from the magnitude of the peaks
knowing the molecular masses and densities of EG and methanol. Once the
standard curves of CH2/OH and CH3/OH proton ratios
versus molar concentration of EG and methanol have been constructed,
one can then use these to calculate the concentrations of EG and methanol in
the larvae from the proton signals generated by the larvae. Specifically, 20
µl of each standard solution was added to 0.2 ml pure D2O
(99.96%) in a 5 mm D2O matched NMR tube. The NMR tube was
preloaded with a sealed glass capillary tube containing chloroform (3.82 mg
CHCl3 or 0.032 mg 1H). Since this internal reference
contained a fixed number of protons, it served as a proton quantity comparison
for all experiments. A blank control sample that contained only 0.2 ml of the
pure D2O and the reference was measured in order to know the
background proton count from residual water in the D2O, and the
data was used for calibration. When calculating the integrated proton peak
area, the value of the chloroform reference was set at 100 and then the
relative area value of each proton peak could be compared among different NMR
spectra so that the proton contributed by the residual H2O in
D2O could be subtracted from the OH proton peak.
The proton NMR experiments were performed on a Mercury 300 NMR spectrometer (Varian, Fort Collins, CO, USA) with proton resonance frequency at 300 MHz and a vertical 7 T magnet, using a single 20° pulse, 5 s relaxation delay and 32 scans for proton Bloch decay acquisition. The data were recorded at 25°C and processed using MestRe-C software (version 2.1.0; Universidad de Santiago de Compostela, Santiago de Compostela, Spain). The data were multiplied by 1 Hz line broadening faction and were Fourier transformed to yield the final spectra. The integrated proton peak area was determined by the MestRe-C software.
Toxicity of 1.5 mol l-1 EG and 1.5 mol l-1
methanol
The 1st instar larvae were immersed in 1.5 mol l-1 EG or 1.5 mol
l-1 methanol for 0 (control), 1, 2, 3, 4, 5, 6 or 8 h in a
centrifuge tube at room temperature (2224°C). After the appropriate
exposure time in the cryoprotectant solutions, approximately 50 of the larvae
were pipetted to a cell strainer to drain the CPA solutions. The strainer was
then blotted with facial tissue and transferred into 5 ml water in a tissue
culture dish for 15 min to wash out the internal CPAs. The number of larvae
was determined under a dissecting microscope and they were then transferred
into 50 ml water in a plastic box. Larvae were fed with slurry comprising a
mixture of tropical fish food (VitaPro Plus Cichlid Power Flakes; M. Reed
Enterprises, Sutter Creek, CA, USA) and active baker's yeast (2% w/v; 2:1 fish
food:baker's yeast in water). The larval feeding and culture methods were
according to the procedures described by Benedict
(1997). After 4 days incubation
in a 26°C incubator, larval survival was assessed on the basis of motility
and normal morphology. The data are expressed as percentage survival relative
to the percentage survival of control untreated larvae.
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Results |
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The signal intensity of each peak (integrated peak area) is proportional to the amount of each proton in the sample. The relative intensities of the CH2 proton peak and the OH proton peak represent the relative molar concentrations of CH2 protons and OH protons, respectively. Therefore, the molar ratio of CH2 protons to OH protons in each standard solution of EG can be measured from its 1H-NMR spectrum. The proton molar ratio (CH2/OH) of each EG solution can be calculated on the basis of the relative molecular mass (62.07) and density (1.11 g ml-1) of pure EG (CH2OHCH2OH). For example, 1.5 mol l-1 EG=93.11 g (1.5x62.07 g), i.e. 83.88 ml (93.11/1.11) EG + 916.12 ml (100083.88 ml), i.e. 50.90 moles (916.12x1/18) water. Each EG molecule has a ratio of four CH2 protons to two OH protons, and each water (HOH) molecule contributes two OH protons. Thus, a 1.5 mol l-1 EG solution contains 6 mol l-1 (4x1.5) CH2 protons and 104.80 mol l-1 [(2x1.5)+(2x50.90)] OH protons. Therefore, proton molar ratio (CH2 protons/OH protons)=6/104.80=5.73%.
According to the above calculations, there exists the following theoretical
relationship between the proton molar ration (percentage), R, and the
molar concentration of EG (CEG):
![]() | (1) |
![]() | (2) |
Fig. 2 compares these calculated proton molar ratios for a series of concentrations of EG and methanol with those obtained experimentally from NMR measurements. The agreement is very close, confirming that the 1H-NMR measurement method is reliable.
|
On the basis of this agreement, one can use measured proton molar ratios to
calculate the concentration of EG and methanol in an unknown sample. This is
achieved by rearranging equations 1 and 2 to derive the following equations:
![]() | (3) |
and
![]() | (4) |
Kinetics of influx and efflux of EG and methanol
The filled circles in Fig.
3A show the measured influx curve for the permeation of EG into
the larvae suspended in 1.5 mol l-1 EG at room temperature. It can
be seen that the larval concentration of EG increased with exposure time
during the first 6 h. At that point, the concentration of EG in the larvae was
1.44 mol l-1, which is 96% of the theoretical maximum if full
equilibration were achieved.
|
In separate experiments, larvae were exposed to 1.5 mol l-1 EG for 4 h, at which time the larval concentration of EG had risen to 1.21 mol l-1, and then transferred to EG-free water for various times before initiating the NMR measurements. The open circles in Fig. 3A show the resulting curve for the efflux of EG. The efflux occurred at a somewhat similar rate as influx, but approximately 0.4 mol l-1 EG remained in larval tissue after 4 h washing.
Fig. 3B shows the results of analogous experiments with methanol. Methanol shows much faster rates of both influx and efflux compared with those of EG. After just a 1 h exposure to 1.5 mol l-1 methanol, the concentration of methanol in the larvae reached a maximum of 1.13 mol l-1, which is only 75% of the theoretical maximum. The internal larval concentration of methanol also appears to drop slowly with permeation times longer than 1 h, but the drop is not statistically significant (P>0.05, N=3). When, after 1 h, the larvae are transferred from methanol to water; the rate of efflux of the methanol (open triangles) is almost as high as the rate of influx.
Toxicity of 1.5 mol l-1 EG and 1.5 mol l-1
methanol
The results of Fig. 3
clearly show that EG and methanol permeate the larvae. The next question is
how high a concentration and exposure time they can tolerate. The toxicity
studies (Fig. 4) show that
larvae can tolerate 1.5 mol l-1 EG and 1.5 mol l-1
methanol at room temperature up to 4 h and 1 h, respectively, with a
normalized survival of 90.6±5.81% and 95.1±2.6%, respectively.
When the exposure time was extended to 5 h in EG and 2 h in methanol, larval
survival dropped significantly (P<0.01 for EG and
P<0.05 for methanol; N=4) when compared with that of
controls. However, larval survival remained rather high (77.9±3.8%)
after 3 h exposure in 1.5 mol l-1 methanol. About 59% of the larvae
survived a 6 h exposure in 1.5 mol l-1 EG, by which time the
concentration of EG within the larvae reached the maximum of 1.44 mol
l-1.
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Discussion |
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Ethylene glycol and methanol were chosen here because EG is the main
permeating CPA used for the cryopreservation of insect eggs
(Mazur et al., 1992a;
Nunamaker and Lockwood, 2001
;
Steponkus et al., 1990
;
Wang et al., 2000
) and because
methanol has been reported to be an effective cryoprotectant in both
slow-cooling (Ali and Shelton,
1993
; Czlonkowska et al.,
1991
; Rall et al.,
1983
,
1984
;
Rapatz, 1973
) and
vitrification (James, 1980
;
Liu et al., 1998
) approaches
to cryopreservation.
Our data indicate that the permeation of EG into larvae is rather slow
(only 37% equilibration in 1h and nearly full equilibration in 6 h), whereas
the permeation of methanol is much faster; the maximum internal concentration
of methanol (75% of equilibrium) being reached in just 1 h. The rates of
efflux of the two CPAs were similar to those for influx. It is not surprising
that methanol permeates rapidly. It has been found to be the only CPA that is
able to penetrate the embryo of zebrafish (Danio rerio;
Hagedorn et al., 1996;
Liu et al., 2001
;
Zhang and Rawson, 1996b
) and
also to penetrate mammalian cells and embryos very rapidly
(Naccache and Sha'afi, 1973
;
Rall et al., 1984
). Although
it permeates rapidly, it seems to be a paradox that the maximum larval tissue
concentration of methanol falls shorter of the theoretical maximum (1.5 mol
l-1) than does that of EG. Other investigators also found that the
proton NMR-measured concentration of CPA in tissues such as cornea, liver or
ovaries does not reach the full theoretical maximum, usually with an
equilibration of 5082% of the theoretical achievable maximum
(Fuller and Busza, 1994
;
Taylor and Busza, 1992
;
Thomas et al., 1997
). Taylor
and Busza (1992
) suggested
that this could be partly due to a fraction of tissue water that is
inaccessible for exchange with CPAs, namely `non-solvent' water, and this
might reflect mitochondrial compartments as proposed by Garlid
(1979
). Thomas et al.
(1997
), however, suggested
another explanation in their work with porcine ovaries; that the tissue
organization could produce an effective diffusion barrier for CPAs. Both of
the two explanations are inapplicable here, however, since EG does permeate
into the larvae to a level of nearly full equilibration. Rather, we think the
explanation here is that significant quantities of the internal methanol in
larval tissues are lost by efflux during the several washes during sample
preparation just before the NMR measurements are initiated. The purpose of the
washing procedure was to remove the surface CPAs with minimal possible loss of
internal CPAs, and it appears to effectively serve this aim for larvae treated
with EG, but less so for the methanol-treated larvae. We also tried using cold
(0°C) H2O and D2O to wash the larvae, considering
that low temperature might slow down the methanol efflux; however, the results
were similar (data not shown).
D2O was used as the medium to finally wash away the residual
water from the larval surface and to suspend the larvae for NMR measurements,
since pure D2O would not contribute any proton signal under proton
NMR. However, commercially pure D2O (99.96%) still contains a
very small amount of H2O and it can be contaminated easily during
the experiments. The residual and contaminated water in D2O could
make a significant difference since a relatively large amount of
D2O was used to suspend the relatively small amount of larvae.
Therefore, a CH3Cl reference was used for all samples in order to
calibrate the data by subtracting the amount of protons from residual
H2O in D2O, the value of which is obtained from a blank
sample of D2O.
Another concern is that larval tissues contain hydroxyl protons from
sources other than water, which also contribute to the OH peak. A pilot
experiment with both fully dehydrated and fully hydrated larvae found that the
OH protons from larval tissues only consist of 0.52% of the total OH protons
of fully hydrated normal larvae. It is a reasonable figure considering that
approximately 82% of the total mass of larvae is water
(Liu and Mazur, in press).
Therefore, the amount of OH protons contributed by larval tissue is
negligible. A final point is that proton exchange occurs between OH (of
H2O, EG or methanol) and OD of D2O to give HOD and
partially deuterated EG or methanol. However, there exists no proton exchange
between CH2 (of EG) or CH3 (of methanol) and OD.
Moreover, once the larvae are in the NMR tube, any proton exchange between OH
and OD will not affect the results of NMR measurements since HOD is visible to
NMR and the resonance frequency of HOD is the same as that of HOH.
The larva is, at a minimum, a three-compartment system consisting of a gut, extracellular tissue space and intracellular space. One important question regarding the CPA concentration in larvae is whether CPA permeation is primarily limited to the gut or whether permeation occurs throughout the larval tissues. While the present study provides no direct answer, it seems unlikely that EG permeation is just confined to the gut since after 6 h exposure the larval EG concentration reaches about 96% of the theoretical maximum, indicating that full equilibration has almost been reached. NMR measured the concentration of CPA in the larvae and not the absolute amounts. Consequently, one possibility is that the larval tissues could have been dehydrated by the EG solution in the gut, thus raising the larval EG concentration in those tissues closer to equilibrium. But we did not see obvious larval shrinkage following up to 12 h exposure in 1.5 mol l-1 EG at room temperature based on microscope observation. Our recent differential scanning calorimetry data on the amount of freezable water (X.-H. Liu and P. Mazur, unpublished data) also indicate that after 46 h exposure in 1.5 mol l-1 EG, the larvae contain a similar amount of freezable water to that of untreated control larvae, suggesting no significant larval dehydration. Therefore, it seems to be the case that EG does fully permeate into the Anopheles larvae following approximately 6 h exposure. Finally, we need to point out that, from the viewpoint of cryopreservation, the concentration of CPA is more important than the absolute amount.
What is the mechanism of CPA permeation? The outer surface of mosquito
larvae is covered by a chitinprotein cuticle, which is presumably not
permeable to CPAs. However, Anopheles larvae are freshwater species
and they possess osmoregulatory mechanisms
(Bradley, 1987). In
hyperosmotic medium, the larvae tend to lose water due to dehydration by the
outward diffusion of water down an osmotic gradient, but they maintain normal
volume by constant drinking. Presumably, when the hyperosmotic medium contains
CPAs, these will enter the gut in the course of this drinking. The gut is
separated from the intestinal epithelium and other tissues by an acellular
chitin-containing sheath, the peritrophic matrix membrane (PM). Recently, the
PM of Anopheles mosquitoes has been found to be permeable to
particles of
148 kDa (Edwards and
Jacobs-Lorena, 2000
). Therefore, the PM is presumably permeable to
EG and methanol, both of which are much smaller than 148 kDa. It is also
reasonable to assume that these low-molecular-mass CPAs are able to permeate
through the larval intestinal epithelium, which is a cellular layer. As far as
the cells themselves, a wide variety of types have been found to be permeable
to both EG and methanol. Although this study sheds no direct light on the
exact mechanism of CPA permeation, it probably involves both passive transport
and a physiological mechanism such as that involved in osmoregulation.
EG appears to be less toxic to Anopheles larvae than is methanol
for the same time exposure. However, the permeation of EG is considerably
slower than that of methanol, and, if we consider larval survival after the
tissues are exposed to the two CPAs for the times required to attain
equivalent concentrations, these are similar. For example, the larval survival
is 95% when, after 1 h exposure, the tissue concentration of methanol reaches
1.13 mol l-1, and the survival is about 93% after exposure to 1.5
mol l-1 EG for 3.5 h, at which time the EG concentration in the
larvae rises to the same concentration of 1.1 mol l-1. EG and
methanol have been reported to be less toxic to a variety of cells and tissues
than other commonly used CPAs such as DMSO and glycerol
(Ali and Shelton, 1993;
Pollock et al., 1991
;
Zhang and Rawson, 1996a
). Our
study shows that high percentages (>90%) of Anopheles larvae
survive the exposure in EG and methanol for a sufficient time period that
allows them to permeate up to reasonably high concentrations without excessive
injury. These promising results encourage us to conduct further investigation
on the cryopreservation of these larvae.
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Acknowledgments |
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