
 pseudomonas aeruginosa has become the most common gram - negative bacterial species associated with serious hospital - acquired infections [ 1 , 2].mortality rates outbreaks from p. aeruginosa in burn units can be significantly high ( 60% ) .immediate use of effective antimicrobial therapy for p. aeruginosa bacteremia has been shown to significantly reduce mortality .
however , the intrinsic resistance of p. aeruginosa to many antimicrobial agents and , in addition , the variety of their increasingly recognized acquired resistance mechanisms make their management in the hospital setting problematic . of great concern
is the growing emergence of multidrug - resistant ( mdr ) strains of p. aeruginosa [ 69 ] , and the severity of associated infections .
unfortunately , the development of new antibiotics with activity against gram - negative organisms has not kept pace with the increase in prevalence of multidrug resistant pathogens . to provide clinicians with credible alternative treatments to reduce the increasing human mortality and morbidity associated with the infectious diseases by drug - resistant bacterial pathogens
, there is a compelling need to develop new therapeutic agents that are effective against drug - resistant mutants .
structurally , nubiotics are synthetic nucleotides and oligonucleotides with nuclease - resistant backbones , and are fully protonated .
although the exact mechanisms of antibiotic actions for nubiotics are not totally understood , they are thought to be indeed different from that of the traditional antimicrobial drugs and the protonated structures are believed , in part , to make nubiotics to be taken up by bacterial cells more easily . as a family , nubiotics has many members , including nu-2 , nu-3 , nu-4 , and nu-5 , which have been shown to have effective therapeutic effect in the burn wound pseudomonas aeruginosa infection model established in mice . nu-3
[ butyl - phosphate-5-thymidine-3-phosphate - butyl ] is a potent nubiotics that is a fully protonated deoxynucleotide .
the therapeutic efficacy of nu-3 against pulmonary pseudomonas aeruginosa infection was evaluated in vivo animal model ,   in which liposome - encapsulated nu-3 presented efficacious antibacterial activity .           to further assess nu-3 as a potential new antibiotic for topical uses , we performed studies for its acute , multiple - dose dermal toxicity and the irritation to skin and eyes and genetic toxicity .
these studies have revealed that nu-3 has hypotoxicity at high dosage , and it has no chronic toxicity and irritation .
this study provides solid data to warrant further development of nubiotics as a novel class of antimicrobial agents for treating pseudomonas aeruginosa infection .
balb / c mice and sprague - dawley rats used in this study were purchased from beijing vital river company .
the body weight ranged from 1720  g for mice and 250300  g for rats .
all the test animals were randomized into groups after being quarantined for 35 days before experiments .
randomly , the body weight variation was within 20% of the mean weight in each sex .
all the mice and rats were housed in a room with a temperature of 2025c , a relative humidity of 40%70% , and a 12-hour - light / dark cycle .
the use and care of the animals followed the regulations for the administration of affairs concerning experimental animals in china ( 11 - 14 - 1988 ) .
chinese hamster ovary cells ( cho and cho - k1 ) were purchased from cell culture center , peking union medical university .
cho - k1 cells were grown in dmem / f-12 supplemented with 10% fetal bovine serum ( fbs ) , 2  mm l - glutamine , and penicillin - streptomycin ( 100  units / ml , 100  g / ml ) .
mice were intragastrically administered with a total dose of 2500 , 1988 , 1580 , 1256 , and 1000  mg / kg / mouse of nu-3 in saline ( table 1 ) .
mice were fasted 24  h before given the test materials and were allowed to get food and water adlibitum after intragastric administration .
animals were observed for their death and subjected to a gross necropsy immediately after they died until day 8 , after which the survivor were euthanized and subjected to a gross necropsy and histopathology observation for toxicity .
livers were removed and fixed by 4% neutral formalin at room temperature for 48  h. the serial tissue sections at 5-m - thick were obtained after embedded in paraffin . each slide was stained with hematoxylin and eosin ( h&e ) then examined under light microscopy ( olympus cx31 ) . throughout the study , the clinical symptoms , neurological behavior and body weight were recorded daily .
three mice of control , male and female group were sacrificed and their lymphocyte suspensions were prepared from the spleen on 24  h after being intragastrically administered with a single dose of nu-3 at 100  mg / kg / mouse .
single - lymphocyte suspensions were incubated in triplicates in 96-well plates at 2    10  cells / well , in rpmi-1640 plus 5% of fetal calf serum ( fcs ) at 37c in a 5% co2 incubator and stimulated for 48  h with 2  g / ml precoated anti - cd3 and 1  g / ml anti - cd28 ( positive control ) , 2  g / ml of bsa ( irrelevant antigen ) , or no antigen ( negative control ) .
t - cell proliferation was evaluated by cell titer 96 aqueous nonradioactive cell proliferation assay was performed according to the manufacturer 's instruction ( promega , usa ) .
the mixture of mts / pms ( 20  l each well ) was added to each well to develop the color .
after 4  h of incubation , the od values of plates were read at 490  nm by a plate reader ( magellan , tecan austria gmbh ) .
data were expressed as stimulation index ( si ) , calculated as the mean reading of triplicate wells of antigen stimulation , and divided by the mean reading of triplicate wells of negative control .
there were ten rats in the treatment group ( 5 males ; 5 females ) in which rats were treated with 50  l of 100  mg / ml nu-3 in saline .
fur was clipped from dorsal area of the trunk 24  h prior to the test and was reclipped as necessary to allow observation and dosing .
the test materials were applied on the shaved area and kept in contact with the help of nonirritating styptic plaster and porous gauze bandage . to ensure that local application of nu-3 remained in the dorsum of animals , the measure of fastening multiply was taken .
briefly , a band - aid was put on the liniment area , and then covered a pad of medical gauze , followed by wrapping up with gauze bandage .
rats were treated with the test materials for 6  h per day . at the end of 6 hours
, any residual material was gently removed from the treatment site using cotton bud soaked in distilled water .
cage side observation was made daily , which included evaluation of the effect on skin , fur , eyes , and respiratory and neural behavior . by the 29th day half of the rats
the remaining animals were observed for further 14 days , and then they were euthanized , subjected to a gross necropsy and organ weights recorded on day 43 . during the study , body weight ,
ten rats were used in the test . on the day prior to dosing , an area on the dorsal surface of the skin was clipped free of hair . on the day of dosing ,
then 10  mg / ml nu-3 in saline was applied to the shaved area of five rats and the skin was covered with nonirritating styptic plaster and porous gauze bandage .
after 6  h of exposure period , the coverings were removed and the test area was rinsed with distilled water .
the rats were examined for the presence of erythema and oedema according to a skin irritation scoring standard at the intervals of 1 , 24 , 48 , and 72  h . rat eye irritation test was used to evaluate the degree to the mucous irritation of nu-3 .
six rats were used for this test and each animal served as its own control .
0.03  ml nu-3 of 1  mg / ml concentration with saline was dripped to the left eyes of the rats , and 0.03  ml saline was dripped to the right eyes of the rats as control .
all the measures were taken to ensure that the test materials are dripped into the conjunctival sac of the rats .
the rats were examined for the presence of changes in eyes according to the eye irritation scoring standard at grading intervals of 1 , 4 , 24 , 48 , 72 and 96  h. then sum of scores calculated was classified according to the eye irritation scoring standard .
the purpose of this study was to assess the ability of nu-3 to induce mutations at the hgprt locus in cultured mammalian cells .
cultures were purged of preexisting mutants by growth for 2 days in dmem / f-12 medium supplemented with 10% fbs , 5    10  m thymidine , 1    10  m hypoxanthine , and 3.2    10  m aminopterin .
the test was performed according to the procedure of o'neill and hsie   and o'neill et al .
the cells which were purged of preexisting mutants were treated for 4  h with the test and control materials and then allowed an expression time of 79 days ; the cells were then replated .
after the period of expression of the mutant phenotype , mutant colonies were selected in the following manner .
each culture flask was trypsinized and the cells were seeded in new flasks as follows : ( a ) 3 flasks each with 200 cells and without the purine analogue 6-thioguanine ; ( b ) 3 flasks each with 2    10 cells and with 6-thioguanine ( 10  g / ml ) .
after 8 days , selection was determined by the formation of colonies resistant to the purine analogue .
next , the colonies were fixed with methanol for 15  min and stained with giemsa ( sigma ) for 30  min .
nu-3 was evaluated in the presence and absence of a metabolic activation system derived from rat liver ( s9 ) .
dimethylbenzanthracene was used in the positive control for the activated assay and ethyl methanesulfonate was used in the positive control for the nonactivated assay .
the nu-3 concentrations for the assay ranged from 500 to 2000  g / ml . the colony counts obtained were used to calculate : ( a ) cloning efficiency ( ce ) in absolute values ( number of colonies formed divided by the number of cells seeded ) ; ( b ) the number of mutant colonies observed in each treatment ; ( c ) the absolute ce observed after selection ; ( d ) the mutation frequency ( mf ) ( number of mutant colonies divided by the number of clonable cells which was equal to the number of cells seeded  the absolute ce after selection ) .
nu-3 was evaluated for its ability to induce micronuclei in the bone marrow of balb / c mice according to hayashi et al . .
cyclophosphamide ( pc ) was used as the positive control , and saline was the negative control .
groups of five male and five female balb / c mice were dosed with nu-3 at 250 , 500 , and 1000  mg / kg on 2 consecutive days via intragastric administration .
all the mice were euthanized 6  h after the last dosing , and the bone marrow was taken to make smear .
bone marrow cells from animals were analyzed for the number of polychromatic erythrocytes ( pces ) that contained at least one micronucleus .
a minimum of 2000 pces was analyzed for the negative control , the positive control and for mice treated with the nu-3 .
the pce fraction was determined by counting a minimum of 200 erythrocytes ( pces plus normochromatic erythrocytes [ nces ] ) .
data were expressed as means    sd and subjected to one - way anova with factors of treatment , genotype or wild type .
mice were intragastrically administered with a single dose of nu-3 at 2500 ( group 1 ) , 1988 ( group 2 ) , 1580 ( group 3 ) , 1256 ( group 4 ) , and 1000 ( group 5 )  mg / kg / mouse ( table 1 ) .
following the single dose , five male and four female mice in group 1 , four female in group 2 died in 24 hours .
one female in group 2 and one female in group 4 died on day 1 . from day 2 to day 8 none of the mice in all the groups died . according to the mortality of each group , the minimum lethal dose ( mld ) was calculated to be 1256  mg / kg . clinical observations of the dying mice attributed to the administration of nu-3 were lethargy , tachypnea , and tremor and disheveled clothing hair .
there were no treatment - related effects on mouse behavior in the cage and body weight for males or females ( data not shown ) . in the early dead mice ,
some pathological changes were found , including livers turning white and brittle with some yellowish brown nidi , the change of the spleen surface into uneven with some hemorrhagic spots , and kidneys showing some gray - white focus on surfaces .
it was also observed that there were some damages and hemorrhage in gastric walls and intestinal tracts of mice which died in 5 hours after being administered .
there was no obvious pathological change at gross necropsy and histopathological changes in liver for scheduled euthanasia mice in the study ( figure 2 ) . to examine
if the administration of nu-3 could affect on immune system , the spleen cells were used to test t - cell proliferation . as shown in figure 3 ,
the treatment with nu-3 had no impact on proliferation of spleen t - cells stimulated with either anti - cd28 ( positive control ) or bsa ( irrelevant antigen ) compared with the cells from the nontreated mice .
rats were applied with 50  l of 100  mg / ml nu-3 ( total dose of 250  mg / kg / day ) for 28 days .
there were no evident toxic symptoms in both treated and control rats during observation time .
control and test rats showed a similar pattern of weight growth , food and water uptake ( figure 4 ) .
although there seemed to be some lagging in water uptake in treated group compared to control ( figure 4(b ) ) , the body weight of test group seemed to increase faster ( figure 4(a ) ) .
there were no treatment - related changes on food uptake in treated rats compared to the controls ( figure 4(c ) ) . during the observation time after administrating nu-3 on the back of the body for 28 days , there were no significant differences between the treated and control rats in the body weight gain although the treated rats took less water and food compared to the controls .
no obvious pathological changes were found in all organs by gross appearance . moreover , there was no significant difference ( p  >  .05 ) in the organ weight between the test groups and the control group ( table 2 ) .
the results of histopathological analysis of necropsy samples on liver , kidney , spleen and skin of the body back where nu-3 was applied showed no significant pathological changes and toxic effects except that there was a slight increase in skin labrocytes of the treated rats in comparison with the controls ' .
hematology and selected serum chemistry analyses also showed no significant differences ( p  >  .05 ) between control and treated groups ( table 3 ) .
after applied with nu-3 on the dorsal surface , the rats were examined for the presence of erythema and oedema on the test area according to a skin irritation scoring standard .
the results showed that the scores for erythema and oedema were 0 at 1 , 24 , 48 , and 72  h after administration , which was the same to the controls ' .
the eye irritation scores were recorded according to the eye irritation scoring standard   at 1 , 4 , 24 , 48 , 72 , and 96  h after administration , all of which were shown to be 0 . therefore , according to the skin and eye irritation scoring standard , all the rats did n't show any toxic symptom after being given nu-3 .
the treated skins and eyes were normal compared to the controls . the mutation frequency was assessed based on the number of mutants per 1    10 clonable cells ( table 4 ) . in the activated assay ,
the mean mutation frequency was 3.3 for the saline negative control , 244 for the dimethylbenzanthracene positive control , 5.4 for the 500  g / ml concentration of nu-3 ; 4.5 for the 1000  g / ml concentration of nu-3 and 6.9 for the 2000  g / ml concentration of nu-3 .
in the nonactivated assay , the mean mutation frequency was 3.3 for the saline negative control ; 187 for the ethylmethanesulfonate positive control , 4.5 for the 500 g / ml concentration of nu-3 , 5.7 for the 1000  g / ml concentration of nu-3 and 8.3 for the 2000  g / ml concentration of nu-3 .
these data suggested that nu-3 was not mutagenic in the testing system both in the activated and nonactivated assays in the dose range from 01000  g / ml .
however , at a very high concentration ( 2000  g / ml ) there appeared to have an increase in mutation frequency , which suggested a weak positive response .
the percentage of pce and nce of the erythrocytic lines in the nu-3-treated groups were not significantly different from that in the control group .
there was a significant difference in the numbers of micronucleated pce between the positive control group and all nu-3 treated groups ( p  <  .01 ) and between the high dosing group and the negative control ( p  <  .03 ) . however , there was no significant difference between the two low - dosing groups and the negative control group ( p  >  .05 ) .
in all the test groups , the pce numbers were almost same between the males and females .
these results indicated that at low and medium dosages , nu-3 caused no evident cytotoxicity . at a very high dose ( 1000  mg / kg ) ,
an increase in the pces with absolute values outside the normal control levels ( i.e. , 0.3%0.4% ) was observed , suggestive of a weak genotoxic potential for nu-3 .
antibiotic drug development has failed to keep pace with the numbers of emerging antibiotic resistant strains and variants .
consequently , the past decade has seen the emergence of the widely publicized health crisis where the means to combat drug - resistant , plague bacteria and viral pandemics are failing .
nu-3 is a novel small molecule and has been examined in several studies for its antibacterial and therapeutic action , showing that it was as effective as intravenously administered ciprofloxacin , a potent and broadspectrum fluoroquinolone .
, we reported that nu-3 is hypotoxical by intragastric administration . in the repeated dermal toxicity test
the irritation study on cutis and mucosa revealed that nu-3 did not irritate the skin tissue .
further studies demonstrated that nu-3 had no notable genetic toxicity both in vitro and in vivo .
taken together , our data suggested that nu-3 is a well tolerated and exhibited no toxicity when administrated via intragastric and topical routes .
chemically , nu-3 is a modified nucleotide , which could potentially cause genetic mutations in the target tissues through disrupting dna or rna replication / transcription . to examine
if this is the case for nu-3 , the golden standard assays in cells and animals were performed and found no meaningful mutational effect on the testing subjects .
it is unlikely that nu-3 could compete with dttp in the cellular environment to be incorporated into dna during replication .
second , although the antibacterial activities of nu-3 have been demonstrated both in vitro and in vivo , its mechanism of action is still not clear .
one of the possible mechanisms for nu-3 action has recently been proposed , which was supported by the preliminary data showing that nu-3 caused significant changes of the membrane polarity .
lastly , the pharmacological data indicated that the bioavailability and pk profile of nu-3 were similar to those from other nucleoside drugs , which supports the notion of its hypotoxicity for pharmaceutical use ( unpublished data ) .
thus , we suggest that the lack of genotoxicity of nu-3 observed in this study may be due to its unique feature of the mechanisms of action .
 nu-3 [ butyl - phosphate-5-thymidine-3-phosphate - butyl ] is a modified nucleotide that has been shown to have antimicrobial activity against a range of bacteria including pseudomonas aeruginosa . 
 however , data on the toxicological profile of nu-3 are still lacking . in the present study , 
 the toxicity of nu-3 was evaluated by the following studies : acute oral toxicity , dermal and mucous membrane irritation , multiple - dose toxicity and genotoxicity in vivo and vitro . 
 the acute oral toxicity test in mice showed that nu-3 had an ld50 of 2001mg / kg body weight . 
 the irritation tests on rats revealed that nu-3 was not irritant , with an irritation scoring of 0 . 
 the multiple - dose toxicity study in rats showed that nu-3 did not cause significant changes in histology , selected serum chemistry , and hematological parameters compared to the controls . 
 rats administrated with multiple - doses of nu-3 showed no visible toxic symptoms . both in vitro and in vivo , nu-3 exhibited no notable genetic toxicity . 
 overall , the data suggest that nu-3 is hypotoxic or nontoxic antimicrobial compound that warrants being further developed for treating pseudomonas aeruginosa infection . 